Crispr cascade assay

ABSTRACT

The present disclosure describes a CRISPR nuclease cascade assay that can detect one or more target nucleic acids of interest of interest at attamolar (aM) (or lower) limits in about 10 minutes or less without the need for amplifying the target nucleic acids of interest. The CRISPR cascade assays utilize signal amplification mechanisms comprising various components including CRISPR nucleases, guide RNAs (gRNAs), blocked nucleic acid molecules, blocked primer molecules, and reporter moieties.

RELATED APPLICATIONS

This application claims priority to U.S. Ser. No. 63/220,987, filed 12 Jul. 2021, and U.S. Ser. No. 63/289,112, filed 13 Dec. 2021.

FIELD OF THE INVENTION

The present disclosure relates to methods, compositions of matter and assay systems used to detect one or more target nucleic acids of interest in a sample. The assay systems provide signal amplification upon detection of a target nucleic acids of interest without amplification of the target nucleic acids.

BACKGROUND OF THE INVENTION

In the following discussion certain articles and methods will be described for background and introductory purposes. Nothing contained herein is to be construed as an “admission” of prior art. Applicant expressly reserves the right to demonstrate, where appropriate, that the articles and methods referenced herein do not constitute prior art under the applicable statutory provisions.

Rapid and accurate identification of infectious agents is important in order to select correct treatment and prevent further spreading of viral infections and pandemic diseases. For example, viral pathogens, such as SARS-CoV-2, and the associated COVID-19 disease require immediate detection and response to decrease mortality, morbidity and transmission.

Classic nucleic acid-guided nuclease or CRISPR (clustered regularly interspaced short palindromic repeats) detection methods usually rely on pre-amplification of target nucleic acids of interest to enhance detection sensitivity. However, amplification increases time to detection and may cause changes to the relative proportion of nucleic acids in samples that, in turn, lead to artifacts or inaccurate results. Improved technologies that allow very rapid and accurate detection of pathogens are therefore needed for timely diagnosis, prevention and treatment of disease, as well as in other applications.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will be apparent from the following written Detailed Description including those aspects illustrated in the accompanying drawings and defined in the appended claims. Further, all of the functionalities described in connection with one embodiment of the compositions and methods described herein are intended to be applicable to the additional embodiments of the compositions and methods described herein except where expressly stated or where the feature or function is incompatible with the additional embodiments. For example, where a given feature or function is expressly described in connection with one embodiment but not expressly mentioned in connection with an alternative embodiment, it should be understood that the feature or function may be deployed, utilized, or implemented in connection with the alternative embodiment unless the feature or function is incompatible with the alternative embodiment.

The present disclosure provides compositions of matter, methods, and cascade assays to detect target nucleic acids of interest. The cascade assays described herein comprise two different ribonucleoprotein complexes and either blocked nucleic acid molecules or blocked primer molecules. The blocked nucleic acid molecules or blocked primer molecules keep one of the ribonucleoprotein complexes “locked” unless and until a target nucleic acid of interest activates the other ribonucleoprotein complex. The present nucleic acid-guided nuclease cascade assay can detect one or more target nucleic acids of interest (e.g., DNA, RNA and/or cDNA) at attamolar (aM) (or lower) limits in about 10 minutes or less without the need for amplifying the target nucleic acid(s) of interest, thereby avoiding the drawbacks of multiplex amplification, such as primer-dimerization. A particularly advantageous feature of the cascade assay is that, with the exception of the gRNA in RNP1, the cascade assay components stay the same no matter what target nucleic acid(s) of interest are being detected. In this sense, the cascade assay is modular.

There is provided herein in one embodiment of the disclosure a reaction mixture comprising: (i) a first ribonucleoprotein (RNP) complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA); wherein the first gRNA comprises a sequence complementary to a target nucleic acid of interest, and wherein the first nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; (ii) a second ribonucleoprotein complex (RNP2) comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest; wherein the second nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; and (iii) a plurality of blocked nucleic acid molecules comprising a sequence complementary to the second gRNA, wherein the blocked nucleic acid molecules cannot activate the RNP1 or the RNP2.

There is provided in a second embodiment of the disclosure, a reaction mixture comprising: (i) a first complex comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA); wherein the first gRNA comprises a sequence complementary to a target nucleic acid of interest, and wherein the first nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; (ii) a second complex comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest; wherein the second nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; and (iii) a plurality of blocked nucleic acid molecules comprising a sequence complementary to the second gRNA, wherein the blocked nucleic acid molecule cannot activate the first or second complex.

Provided in a third embodiment is a reaction mixture comprising: (i) a first ribonucleoprotein (RNP) (RNP1) complex comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA); wherein the first gRNA comprises a sequence complementary to a target nucleic acid of interest, and wherein the first nucleic acid-guided nuclease exhibits both sequence-specific activity and non-sequence-specific activity; (ii) a second ribonucleoprotein (RNP2) complex comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest; wherein the second nucleic acid-guided nuclease exhibits both sequence-specific activity and non-sequence-specific activity; and (iii) a plurality of blocked nucleic acid molecules comprising a sequence complementary to the second gRNA, wherein the blocked nucleic acid molecules do not bind to the RNP1 complex or the RNP2 complex. In yet another fourth embodiment of the disclosure there is provided a reaction mixture comprising: (i) a first complex comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA); wherein the first gRNA comprises a sequence complementary to a target nucleic acid of interest, and wherein the first nucleic acid-guided nuclease exhibits both sequence-specific activity and non-sequence-specific activity; (ii) a second complex comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest; wherein the second nucleic acid-guided nuclease exhibits both sequence-specific activity and non-sequence-specific activity; and (iii) a plurality of blocked nucleic acid molecules comprising a sequence complementary to the second gRNA, wherein the blocked nucleic acid molecules are not recognized by the RNP1s or RNP2s.

A fifth embodiment provides a cascade assay method for detecting a target nucleic acid of interest in a sample comprising the steps of: (a) providing a reaction mixture comprising: (i) a first ribonucleoprotein (RNP) complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA); wherein the first gRNA comprises a sequence complementary to a target nucleic acid of interest, and wherein the first nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; (ii) a second ribonucleoprotein complex (RNP2) comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest; wherein the second nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; and (iii) a plurality of blocked nucleic acid molecules comprising a sequence complementary to the second gRNA, wherein the blocked nucleic acid molecules cannot activate the RNP1 or the RNP2; (b) contacting the reaction mixture with the sample under conditions that allow the target nucleic acid of interest in the sample to bind to RNP1; wherein upon binding of the target nucleic acid of interest RNP1 becomes active initiating trans-cleavage of at least one of the blocked nucleic acid molecules thereby producing at least one unblocked nucleic acid molecule and the at least one unblocked nucleic acid molecule binds to RNP2 initiating cleavage of at least one further blocked nucleic acid molecule; and (c) detecting products of the cleavage, thereby detecting the target nucleic acid of interest in the sample.

In a sixth embodiment there is provided a kit for detecting a target nucleic acid of interest in a sample comprising: (i) a first ribonucleoprotein (RNP1) complex (RNP1) comprising a first nucleic acid-guided nuclease and a first gRNA, wherein the first gRNA comprises a sequence complementary to the target nucleic acid of interest; and wherein binding of the RNP1 complex to the target nucleic acid of interest activates cis-cleavage and trans-cleavage activity of the first nucleic acid-guided nuclease; (ii) a second ribonucleoprotein complex (RNP2) comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest; a (iii) plurality of blocked nucleic acid molecules comprising a sequence corresponding to the second gRNA, wherein trans-cleavage activity of the blocked nucleic acid molecules results in at least one unblocked nucleic acid molecule; and wherein the unblocked nucleic acid molecule activates trans-cleavage activity of the second nucleic acid-guided nuclease in at least one RNP2 initiating cleavage of more blocked nucleic acid molecules; and (iv) a reporter moiety, wherein the reporter molecule comprises a nucleic acid molecule and/or is operably linked to the blocked nucleic acid molecule and produces a detectable signal upon trans-cleavage activity by the RNP1 or the RNP2, to identify the presence of the target nucleic acid of interest in the sample.

In some aspects of any one of the aforementioned embodiments, the first and/or second nucleic acid-guided nuclease is a Cas3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas14, Cas12h, Cas12i, Cas12j, Cas13a, Cas13b nuclease; in some aspects, the first nucleic acid-guided nuclease can is a different nucleic acid-guided nuclease than the second nucleic acid-guided nuclease; in some aspects, the first and/or second nucleic acid-guided nuclease is a Type V nucleic acid-guided nuclease or a Type VI nucleic acid-guided nuclease and/or in some aspects, the first and/or second nucleic acid-guided nuclease comprises a RuvC nuclease domain or a RuvC-like nuclease domain and lacks an HNH nuclease domain.

In some aspects of any one of the aforementioned embodiments, the blocked nucleic acid molecules comprise a structure represented by any one of Formulas I-IV, wherein Formulas I-IV comprise in the 5′-to-3′ direction:

(a)A-(B-L)_(J)-C-M-T-D  (Formula I);

-   -   wherein A is 0-15 nucleotides in length;     -   B is 4-12 nucleotides in length;     -   L is 3-25 nucleotides in length;     -   J is an integer between 1 and 10;     -   C is 4-15 nucleotides in length;     -   M is 1-25 nucleotides in length or is absent, wherein if M is         absent then A-(B-L)_(J)-C and T-D are separate nucleic acid         strands;     -   T is 17-135 nucleotides in length and comprises at least 50%         sequence complementarity to B and C; and     -   D is 0-10 nucleotides in length and comprises at least 50%         sequence complementarity to A;

(b)D-T-T′-C-(L-B)_(J)-A  (Formula II);

-   -   wherein D is 0-10 nucleotides in length;     -   T-T′ is 17-135 nucleotides in length;     -   T′ is 1-10 nucleotides in length and does not hybridize with T;     -   C is 4-15 nucleotides in length and comprises at least 50%         sequence complementarity to T;     -   L is 3-25 nucleotides in length and does not hybridize with T;     -   B is 4-12 nucleotides in length and comprises at least 50%         sequence complementarity to T;     -   J is an integer between 1 and 10;     -   A is 0-15 nucleotides in length and comprises at least 50%         sequence complementarity to D;

(c)T-D-M-A-(B-L)_(J)-C  (Formula III);

-   -   wherein T is 17-135 nucleotides in length;     -   D is 0-10 nucleotides in length;     -   M is 1-25 nucleotides in length or is absent, wherein if M is         absent then T-D and A-(B-L)_(J)-C are separate nucleic acid         strands;     -   A is 0-15 nucleotides in length and comprises at least 50%         sequence complementarity to D;     -   B is 4-12 nucleotides in length and comprises at least 50%         sequence complementarity to T;     -   L is 3-25 nucleotides in length;     -   J is an integer between 1 and 10; and     -   C is 4-15 nucleotides in length; or

(d)T-D-M-A-L_(p)-C  (Formula IV);

-   -   wherein T is 17-31 nucleotides in length (e.g., 17-100, 17-50,         or 17-25);     -   D is 0-15 nucleotides in length;     -   M is 1-25 nucleotides in length;     -   A is 0-15 nucleotides in length and comprises a sequence         complementary to D; and     -   L is 3-25 nucleotides in length;     -   p is 0 or 1;     -   C is 4-15 nucleotides in length and comprises a sequence         complementary to T.

And in Some Aspects,

-   -   (a) T of Formula I comprises at least 80% sequence         complementarity to B and C;     -   (b) D of Formula I comprises at least 80% sequence         complementarity to A;     -   (c) C of Formula II comprises at least 80% sequence         complementarity to T;     -   (d) B of Formula II comprises at least 80% sequence         complementarity to T;     -   (e) A of Formula II comprises at least 80% sequence         complementarity to D;     -   (f) A of Formula III comprises at least 80% sequence         complementarity to D;     -   (g) B of Formula III comprises at least 80% sequence         complementarity to T;     -   (h) A of Formula IV comprises at least 80% sequence         complementarity to D; and/or     -   (i) C of Formula IV comprises at least 80% sequence         complementarity to T.

In some aspects of the aforementioned embodiments, the blocked nucleic acid molecules comprise a first sequence complementary to the second gRNA and a second sequence not complementary to the second gRNA, wherein the second sequence at least partially hybridizes to the first sequence resulting in at least one loop.

In some aspects of the aforementioned embodiments, the reaction mixture comprises about 1 fM to about 10 μM of the RNP1 and in some aspects the reaction mixture comprises about 1 fM to about 1 mM of the RNP2.

In some aspects of the aforementioned embodiments, the reaction mixture comprises at least two different RNP1s, wherein different RNP1s comprise different gRNA sequences, and in some aspects the reaction mixture comprises 2 to 10000 different RNP1s, or 2 to 1000 different RNP1s, or 2 to 100 different RNP1s, or 2 to 10 different RNP1s.

In some aspects of the aforementioned embodiments, the blocked nucleic acid molecules include the sequence of any one of SEQ ID NOs: 14-1421.

In some aspects of the aforementioned embodiments, the blocked nucleic acid molecules are circular, and in some aspects the blocked nucleic acid molecules are linear.

In some aspects the K_(d) of the blocked nucleic acid molecules to the RNP2 is about 10⁵-fold greater, 10⁶-fold greater, 10⁷-fold greater, 10⁸-fold greater, 10⁹-fold greater, 10¹⁰-fold greater or more than the K_(d) of unblocked nucleic acid molecules.

In some aspects of the aforementioned embodiments, the target nucleic acid of interest is of bacterial, viral, fungal, mammalian or plant origin, and in some aspects, the sample may include peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, and umbilical cord blood; food; agricultural products; pharmaceuticals; cosmetics, nutraceuticals; personal care products; environmental substances such as soil, water, or air; industrial sites and products; or manufactured or natural chemicals and compounds.

In some aspects of the aforementioned embodiments, the reaction mixture further comprises a reporter moiety: wherein the reporter moiety comprises a DNA, RNA or chimeric nucleic acid molecule that is operably linked to the blocked nucleic acid molecule and produces a detectable signal upon cleavage by RNP1 and/or RNP2; or wherein the reporter moiety comprises a DNA, RNA or chimeric nucleic acid molecule and is not operably linked to the blocked nucleic acid molecule and produces a detectable signal upon cleavage by RNP1 and/or RNP2. In some aspects, the detectable signal is produced within about 1-10 minutes upon binding of the target nucleic acid of interest to RNP1; in some aspects, the detectable signal is a fluorescent, chemiluminescent, radioactive, colorimetric or optical signal; and/or in some aspects, the reporter moiety comprises a modified nucleoside or nucleotide including but not limited to locked nucleic acids (LNAs), peptide nucleic acids (PNAs), 2′-O-methyl (2′-O-Me) modified nucleosides, 2′-fluoro (2′-F) modified nucleoside, and/or a phosphorothioate (PS) bonds.

In some aspects of the aforementioned embodiments, the blocked nucleic acid molecules do not comprise a PAM sequence, yet in other aspects, the blocked nucleic acid molecules comprise a PAM sequence, and in some aspects the PAM sequence is disposed between the first and second sequences, wherein the first sequence is 5′ to the PAM sequence.

In some aspects of the aforementioned embodiments, the blocked nucleic acid molecule is a blocked primer molecule.

In a seventh embodiment of the disclosure, there is provided a blocked nucleic acid molecule comprising: a first region recognized by a ribonucleoprotein (RNP) complex; one or more second regions not complementary to the first region; and one or more third regions complementary and hybridized to the first region, wherein cleavage of the one or more second regions results in dehybridization of the third region from the first region, resulting in an unblocked nucleic acid molecule.

An eighth embodiment provides a method of unblocking a blocked nucleic acid comprising: (a) providing a blocked nucleic acid molecule comprising: a first region recognized by a ribonucleoprotein (RNP) complex; one or more second regions not complementary to the first region; and one or more third regions complementary and hybridized to the first region, wherein cleavage of the one or more second regions results in dehybridization of the third region from the first region, resulting in an unblocked nucleic acid molecule; and (b) initiating cleavage of the one or more second regions, wherein the blocked nucleic acid molecule becomes an unblocked nucleic acid molecule.

A ninth embodiment provides a composition of matter comprising: a first region recognized by a ribonucleoprotein (RNP) complex; one or more second regions of not complementary to the first region; and one or more third regions complementary and hybridized to the first region, wherein cleavage of the one or more second regions results in dehybridization of the third region from the first region, resulting in an unblocked nucleic acid molecule; and the RNP complex comprising a gRNA that is complementary to the first region and a nucleic acid-guided nuclease, wherein the nucleic acid-guided nuclease exhibits both sequence-specific and non-sequence-specific nuclease activity.

Additionally, a tenth embodiment of the disclosure provides a cascade assay method of detecting a target nucleic acid of interest in a sample comprising the steps of: (a) providing a reaction mixture comprising: (i) a first ribonucleoprotein (RNP) complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA); wherein the first gRNA comprises a sequence complementary to a target nucleic acid of interest, and wherein the first nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; (ii) a second ribonucleoprotein complex (RNP2) comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest; wherein the second nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; and (iii) a plurality of linear blocked nucleic acid molecules comprising a sequence complementary to the second gRNA, wherein the linear blocked nucleic acid molecules cannot activate the RNP1 or the RNP2; (b) contacting the reaction mixture with the sample under conditions that allow the target nucleic acid of interest in the sample to bind to RNP1; wherein upon binding of the target nucleic acid of interest RNP1 becomes active initiating trans-cleavage of at least one of the linear blocked nucleic acid molecules thereby producing at least one linear unblocked nucleic acid molecule and the at least one linear unblocked nucleic acid molecule to RNP2 initiating cleavage of at least one further linear blocked nucleic acid molecule; and (c) detecting the cleavage products, thereby detecting the target nucleic acid of interest in the sample.

In some aspects of any one of the aforementioned embodiments, the first and/or second nucleic acid-guided nuclease is a Cas3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas14, Cas12h, Cas12i, Cas12j, Cas13a, Cas13b nuclease; in some aspects, the first nucleic acid-guided nuclease can is a different nucleic acid-guided nuclease than the second nucleic acid-guided nuclease; in some aspects, the first and/or second nucleic acid-guided nuclease is a Type V nucleic acid-guided nuclease or a Type VI nucleic acid-guided nuclease and/or in some aspects, the first and/or second nucleic acid-guided nuclease comprises a RuvC nuclease domain or a RuvC-like nuclease domain and lacks an HNH nuclease domain.

In some aspects, the blocked nucleic acid molecule comprises a structure represented by any one of Formulas I-IV, wherein Formulas I-IV are in the 5′-to-3′ direction:

(a)A-(B-L)_(J)-C-M-T-D  (Formula I);

-   -   wherein A is 0-15 nucleotides in length;     -   B is 4-12 nucleotides in length;     -   L is 3-25 nucleotides in length;     -   J is an integer between 1 and 10;     -   C is 4-15 nucleotides in length;     -   M is 1-25 nucleotides in length or is absent, wherein if M is         absent then A-(B-L)_(J)-C and T-D are separate nucleic acid         strands;     -   T is 17-135 nucleotides in length and comprises at least 50%         sequence complementarity to B and C; and     -   D is 0-10 nucleotides in length and comprises at least 50%         sequence complementarity to A;

(b)D-T-T′-C-(L-B)_(J)-A  (Formula II);

-   -   wherein D is 0-10 nucleotides in length;     -   T-T′ is 17-135 nucleotides in length;     -   T′ is 1-10 nucleotides in length and does not hybridize with T;     -   C is 4-15 nucleotides in length and comprises at least 50%         sequence complementarity to T;     -   L is 3-25 nucleotides in length and does not hybridize with T;     -   B is 4-12 nucleotides in length and comprises at least 50%         sequence complementarity to T;     -   J is an integer between 1 and 10;     -   A is 0-15 nucleotides in length and comprises at least 50%         sequence complementarity to D;

(c)T-D-M-A-(B-L)_(J)-C  (Formula III);

-   -   wherein T is 17-135 nucleotides in length;     -   D is 0-10 nucleotides in length;     -   M is 1-25 nucleotides in length or is absent, wherein if M is         absent then T-D and A-(B-L)_(J)-C are separate nucleic acid         strands;     -   A is 0-15 nucleotides in length and comprises at least 50%         sequence complementarity to D;     -   B is 4-12 nucleotides in length and comprises at least 50%         sequence complementarity to T;     -   L is 3-25 nucleotides in length;     -   J is an integer between 1 and 10; and     -   C is 4-15 nucleotides in length; or

(d)T-D-M-A-L_(p)-C  (Formula IV);

-   -   wherein T is 17-31 nucleotides in length (e.g., 17-100, 17-50,         or 17-25);     -   D is 0-15 nucleotides in length;     -   M is 1-25 nucleotides in length;     -   A is 0-15 nucleotides in length and comprises a sequence         complementary to D; and     -   L is 3-25 nucleotides in length;     -   p is 0 or 1;     -   C is 4-15 nucleotides in length and comprises a sequence         complementary to T.

Further

-   -   (a) T of Formula I comprises at least 80% sequence         complementarity to B and C;     -   (b) D of Formula I comprises at least 80% sequence         complementarity to A;     -   (c) C of Formula II comprises at least 80% sequence         complementarity to T;     -   (d) B of Formula II comprises at least 80% sequence         complementarity to T;     -   (e) A of Formula II comprises at least 80% sequence         complementarity to D;     -   (f) A of Formula III comprises at least 80% sequence         complementarity to D;     -   (g) B of Formula III comprises at least 80% sequence         complementarity to T;     -   (h) A of Formula IV comprises at least 80% sequence         complementarity to D; and/or     -   (i) C of Formula IV comprises at least 80% sequence         complementarity to T.

In some aspects of the aforementioned embodiments, the blocked nucleic acid molecule comprises a modified nucleoside or nucleotide, including but not limited to a locked nucleic acid (LNA), peptide nucleic acid (PNA), 2′-O-methyl (2′-O-Me) modified nucleoside, 2′-fluoro (2′-F) modified nucleoside, and/or a phosphorothioate (PS) bond. In some aspects, the blocked nucleic acid molecule includes the sequence of any one of SEQ ID NOs: 14-1421; the blocked nucleic acid molecule is a blocked primer molecule; the blocked nucleic acid molecule does not comprise a PAM sequence; and/or in some aspects the blocked nucleic acid molecule comprises a PAM sequence, and the PAM sequence is disposed between the first and second sequences, wherein the first sequence is 5′ to the PAM sequence.

In some aspects of the aforementioned embodiments, the reaction mixture further comprises a reporter moiety: wherein the reporter moiety comprises a DNA, RNA or chimeric nucleic acid molecule that is operably linked to the blocked nucleic acid molecule and produces a detectable signal upon cleavage by RNP1 and/or RNP2; or wherein the reporter moiety comprises a DNA, RNA or chimeric nucleic acid molecule and is not operably linked to the blocked nucleic acid molecule and produces a detectable signal upon cleavage by RNP1 and/or RNP2. In some aspects, the detectable signal is produced within about 1-10 minutes upon binding of the target nucleic acid of interest to RNP1; in some aspects, the detectable signal is a fluorescent, chemiluminescent, radioactive, colorimetric or optical signal; and/or in some aspects, the reporter moiety comprises a modified nucleoside or nucleotide including but not limited to locked nucleic acids (LNAs), peptide nucleic acids (PNAs), 2′-O-methyl (2′-O-Me) modified nucleosides, 2′-fluoro (2′-F) modified nucleoside, and/or a phosphorothioate (PS) bonds.

In some aspects, the K_(d) of the blocked nucleic acid molecules to the RNP2 is about 10⁵-fold greater, 10⁶-fold greater, 10⁷-fold greater, 10⁸-fold greater, 10⁹-fold greater, 10¹⁰-fold greater or more than the K_(d) of unblocked nucleic acid molecules.

In an eleventh embodiment, there is provided composition of matter comprising a ribonucleoprotein (RNP) complex and a blocked nucleic acid molecule, wherein the blocked nucleic acid molecule is represented by any one of Formula I-IV, wherein Formulas I-IV comprise in the 5′-to-3′ direction comprises:

(a)A-(B-L)_(J)-C-M-T-D  (Formula I);

-   -   wherein A is 0-15 nucleotides in length;     -   B is 4-12 nucleotides in length;     -   L is 3-25 nucleotides in length;     -   J is an integer between 1 and 10;     -   C is 4-15 nucleotides in length;     -   M is 1-25 nucleotides in length or is absent, wherein if M is         absent then A-(B-L)_(J)-C and T-D are separate nucleic acid         strands;     -   T is 17-135 nucleotides in length and comprises at least 50%         sequence complementarity to B and C; and     -   D is 0-10 nucleotides in length and comprises at least 50%         sequence complementarity to A;

(b)D-T-T′-C-(L-B)_(J)-A  (Formula II);

-   -   wherein D is 0-10 nucleotides in length;     -   T-T′ is 17-135 nucleotides in length;     -   T′ is 1-10 nucleotides in length and does not hybridize with T;     -   C is 4-15 nucleotides in length and comprises at least 50%         sequence complementarity to T;     -   L is 3-25 nucleotides in length and does not hybridize with T;     -   B is 4-12 nucleotides in length and comprises at least 50%         sequence complementarity to T;     -   J is an integer between 1 and 10;     -   A is 0-15 nucleotides in length and comprises at least 50%         sequence complementarity to D;

(c)T-D-M-A-(B-L)_(J)-C  (Formula III);

-   -   wherein T is 17-135 nucleotides in length;     -   D is 0-10 nucleotides in length;     -   M is 1-25 nucleotides in length or is absent, wherein if M is         absent then T-D and     -   A-(B-L)_(J)-C are separate nucleic acid strands;     -   A is 0-15 nucleotides in length and comprises at least 50%         sequence complementarity to D;     -   B is 4-12 nucleotides in length and comprises at least 50%         sequence complementarity to T;     -   L is 3-25 nucleotides in length;     -   J is an integer between 1 and 10; and     -   C is 4-15 nucleotides in length; or

(d)T-D-M-A-L_(p)-C  (Formula IV);

-   -   wherein T is 17-31 nucleotides in length (e.g., 17-100, 17-50,         or 17-25);     -   D is 0-15 nucleotides in length;     -   M is 1-25 nucleotides in length;     -   A is 0-15 nucleotides in length and comprises a sequence         complementary to D; and     -   L is 3-25 nucleotides in length;     -   p is 0 or 1;     -   C is 4-15 nucleotides in length and comprises a sequence         complementary to T.

In Some Aspects of this Embodiment,

T of Formula I comprises at least 80% sequence complementarity to B and C;

-   -   (a) D of Formula I comprises at least 80% sequence         complementarity to A;     -   (b) C of Formula II comprises at least 80% sequence         complementarity to T;     -   (c) B of Formula II comprises at least 80% sequence         complementarity to T;     -   (d) A of Formula II comprises at least 80% sequence         complementarity to D;     -   (e) A of Formula III comprises at least 80% sequence         complementarity to D;     -   (f) B of Formula III comprises at least 80% sequence         complementarity to T;     -   (g) A of Formula IV comprises at least 80% sequence         complementarity to D; and/or     -   (h) C of Formula IV comprises at least 80% sequence         complementarity to T.

In some aspects of the aforementioned embodiment, the blocked primer molecules include the sequence of any one of SEQ ID NOs: 14-1421.

In some aspects of the aforementioned embodiment, the RNP complex comprises a Cas3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas14, Cas12h, Cas12i, Cas12j, Cas13a, Cas13b nuclease; in some aspects, the RNP complex comprises a Type V nucleic acid-guided nuclease or a Type VI nucleic acid-guided nuclease and/or in some aspects, the RNP complex comprises a RuvC nuclease domain or a RuvC-like nuclease domain and lacks an HNH nuclease domain.

In some aspects of the aforementioned embodiment, the blocked nucleic acid molecule comprises a modified nucleoside or nucleotide comprises a locked nucleic acid (LNA), peptide nucleic acid (PNA), 2′-O-methyl (2′-O-Me) modified nucleoside, 2′-fluoro (2′-F) modified nucleoside, and/or a phosphorothioate (PS) bond.

In some aspects, the blocked nucleic acid molecule does not comprise a PAM sequence, and in other aspects, the blocked nucleic acid molecule comprises a PAM sequence where the PAM sequence is disposed between the first and second sequences, wherein the first sequence is 5′ to the PAM sequence. In some aspects, the blocked nucleic acid molecule is a blocked primer molecule.

In some aspects of the aforementioned embodiment(s), the composition of matter further comprises a reporter moiety: wherein the reporter moiety comprises a DNA, RNA or chimeric nucleic acid molecule that is operably linked to the blocked nucleic acid molecule and produces a detectable signal upon cleavage by RNP1 and/or RNP2; or wherein the reporter moiety comprises a DNA, RNA or chimeric nucleic acid molecule and is not operably linked to the blocked nucleic acid molecule and produces a detectable signal upon cleavage by RNP1 and/or RNP2. In some aspects, the detectable signal is produced within about 1-10 minutes upon binding of the target nucleic acid of interest to RNP1; in some aspects, the detectable signal is a fluorescent, chemiluminescent, radioactive, colorimetric or optical signal; and/or in some aspects, the reporter moiety comprises a modified nucleoside or nucleotide including but not limited to locked nucleic acids (LNAs), peptide nucleic acids (PNAs), 2′-O-methyl (2′-O-Me) modified nucleosides, 2′-fluoro (2′-F) modified nucleoside, and/or a phosphorothioate (PS) bonds.

Yet another embodiment provides a reaction mixture comprising: (i) a first ribonucleoprotein (RNP) complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA); wherein the first gRNA comprises a sequence complementary to a target nucleic acid of interest, and wherein the first nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; (ii) a second ribonucleoprotein complex (RNP2) comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest; wherein the second nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; (iii) a plurality of template molecules comprising a sequence corresponding to the second gRNA; (iv) a plurality of blocked primer molecules comprising a sequence complementary to the template molecules, wherein the blocked nucleic acid molecules cannot be extended by a polymerase; and (v) a polymerase and a plurality of nucleotides.

Another embodiment provides a cascade assay method for detecting a target nucleic acid of interest in a sample comprising: (a) providing a reaction mixture comprising: (i) a first ribonucleoprotein (RNP) complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA); wherein the first gRNA comprises a sequence complementary to a target nucleic acid of interest, and wherein the first nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; (ii) a second ribonucleoprotein complex (RNP2) comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest; wherein the second nucleic acid-guided nuclease exhibits both cis-cleavage activity and trans-cleavage activity; (iii) a plurality of template molecules comprising a sequence corresponding to the second gRNA; (iv) a plurality of blocked primer molecules comprising a sequence complementary to the template molecules, wherein the blocked nucleic acid molecules cannot be extended by a polymerase; and (v) a polymerase and a plurality of nucleotides; (b) contacting the reaction mixture with the sample under conditions that allow target nucleic acids of interest in the sample to bind to the first gRNA, wherein: upon binding of the target nucleic acid of interest, the RNP1 active cleaving at least one of the blocked primer molecules, thereby producing at least one unblocked primer molecule that can be extended by the polymerase; at least one unblocked primer molecule binds to one of the template molecules and is extended by the polymerase and nucleotides to form at least one synthesized activating molecule having a sequence complementary to the second gRNA; at least one synthesized activating molecule binds to the second gRNA, and RNP2 becomes active cleaving at least one further blocked primer molecule; and detecting the cleavage products of step (b), thereby detecting the target nucleic acid of interest in the sample.

In some aspects the K_(d) of the blocked nucleic acid molecules to the RNP2 is about 10⁵-fold greater, 10⁶-fold greater, 10⁷-fold greater, 10⁸-fold greater, 10⁹-fold greater, 10¹⁰-fold greater or more than the K_(d) of unblocked nucleic acid molecules.

A further embodiment provides a kit for detecting a target nucleic acid of interest in a sample comprising: (i) a first ribonucleoprotein complex (RNP1) comprising a first nucleic acid-guided nuclease and a first gRNA, wherein the first gRNA comprises a sequence complementary to the target nucleic acid of interest; and wherein binding of the RNP1 complex to the target nucleic acid of interest activates cis-cleavage and trans-cleavage activity of the first nucleic acid-guided nuclease; (ii) a second ribonucleoprotein complex (RNP2) comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest; (iii) a plurality of template molecules comprising a non-target sequence to the second gRNA; (iv) a polymerase and nucleotides; (v) a plurality of blocked primer molecules comprising a sequence complementary to the template molecules, wherein the blocked primer molecule cannot be extended by the polymerase, trans-cleavage of the blocked primer molecules results in at least one unblocked primer molecule; and wherein the unblocked primer molecule can bind to at least one the template molecule and be extended by the polymerase to form a synthesized activating molecule; and (vi) a reporter moiety, wherein the reporter moiety comprises a nucleic acid molecule and/or is operably linked to the blocked primer molecule and produces a detectable signal upon trans-cleavage activity of the blocked primer molecule by the RNP1 or the RNP2, to identify the presence of the target nucleic acid of interest in the sample.

In any of these embodiments, the first and/or second nucleic acid-guided nuclease in the reaction mixture is a Cas3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas14, Cas12h, Cas12i, Cas12j, Cas13a, Cas13b nuclease; in some aspects, the first nucleic acid-guided nuclease is a different nucleic acid-guided nuclease than the second nucleic acid-guided nuclease; in some aspects the first and/or second nucleic acid-guided nuclease is a Type V nucleic acid-guided nuclease or a Type VI nucleic acid-guided nuclease; and in some aspects, the first and/or second nucleic acid-guided nuclease comprises a RuvC nuclease domain or a RuvC-like nuclease domain and lacks an HNH nuclease domain.

In some aspects the blocked primer molecules comprise a first sequence complementary to the second gRNA and a second sequence not complementary to the second gRNA, wherein the second sequence at least partially hybridizes to the first sequence resulting in at least one loop; and in some aspects, the blocked primer molecules comprise a structure represented by any one of Formulas I-IV, wherein Formulas I-IV are in the 5′-to-3′ direction:

(a)A-(B-L)_(J)-C-M-T-D  (Formula I);

-   -   wherein A is 0-15 nucleotides in length;     -   B is 4-12 nucleotides in length;     -   L is 3-25 nucleotides in length;     -   J is an integer between 1 and 10;     -   C is 4-15 nucleotides in length;     -   M is 1-25 nucleotides in length or is absent, wherein if M is         absent then A-(B-L)_(J)-C and T-D are separate nucleic acid         strands;     -   T is 17-135 nucleotides in length and comprises at least 50%         sequence complementarity to B and C; and     -   D is 0-10 nucleotides in length and comprises at least 50%         sequence complementarity to A;

(b)D-T-T′-C-(L-B)_(J)-A  (Formula II);

-   -   wherein D is 0-10 nucleotides in length;     -   T-T′ is 17-135 nucleotides in length;     -   T′ is 1-10 nucleotides in length and does not hybridize with T;     -   C is 4-15 nucleotides in length and comprises at least 50%         sequence complementarity to T;     -   L is 3-25 nucleotides in length and does not hybridize with T;     -   B is 4-12 nucleotides in length and comprises at least 50%         sequence complementarity to T;     -   J is an integer between 1 and 10;     -   A is 0-15 nucleotides in length and comprises at least 50%         sequence complementarity to D;

(c)T-D-M-A-(B-L)_(J)-C  (Formula III);

-   -   wherein T is 17-135 nucleotides in length;     -   D is 0-10 nucleotides in length;     -   M is 1-25 nucleotides in length or is absent, wherein if M is         absent then T-D and A-(B-L)_(J)-C are separate nucleic acid         strands;     -   A is 0-15 nucleotides in length and comprises at least 50%         sequence complementarity to D;     -   B is 4-12 nucleotides in length and comprises at least 50%         sequence complementarity to T;     -   L is 3-25 nucleotides in length;     -   J is an integer between 1 and 10; and     -   C is 4-15 nucleotides in length; or

(d)T-D-M-A-L_(p)-C  (Formula IV);

-   -   wherein T is 17-31 nucleotides in length (e.g., 17-100, 17-50,         or 17-25);     -   D is 0-15 nucleotides in length;     -   M is 1-25 nucleotides in length;     -   A is 0-15 nucleotides in length and comprises a sequence         complementary to D; and     -   L is 3-25 nucleotides in length;     -   p is 0 or 1;     -   C is 4-15 nucleotides in length and comprises a sequence         complementary to T.

In Some Aspects,

-   -   (a) T of Formula I comprises at least 80% sequence         complementarity to B and C;     -   (b) D of Formula I comprises at least 80% sequence         complementarity to A;     -   (c) C of Formula II comprises at least 80% sequence         complementarity to T;     -   (d) B of Formula II comprises at least 80% sequence         complementarity to T;     -   (e) A of Formula II comprises at least 80% sequence         complementarity to D;     -   (f) A of Formula III comprises at least 80% sequence         complementarity to D;     -   (g) B of Formula III comprises at least 80% sequence         complementarity to T;     -   (h) A of Formula IV comprises at least 80% sequence         complementarity to D; and/or     -   (i) C of Formula IV comprises at least 80% sequence         complementarity to T.

In some aspects the reaction mixture comprises about 1 fM to about 10 μM of the RNP1, and in some aspects, the reaction mixture of claim 1, wherein the reaction mixture comprises about 1 fM to about 1 mM of the RNP2.

In some aspects of these embodiments, the reaction mixture comprises at least two different RNP1s, wherein different RNP1s comprise different gRNA sequences, and in some aspects, the reaction mixture comprises 2 to 10000 different RNP1s, 2 to 1000 different RNP1s, 2 to 100 different RNP1s, or 2 to 10 different RNP1s.

In some aspects the blocked primer molecules include the sequence of any one of SEQ ID NOs: 14-1421. In some aspects the K_(d) of the blocked primer molecules to the RNP2 is about 10⁵-fold greater, 10⁶-fold greater, 10⁷-fold greater, 10⁸-fold greater, 10⁹-fold greater, 10¹⁰-fold greater or more than the K_(d) of unblocked primer molecules.

In some aspects of the aforementioned embodiments, the reaction mixture further comprises a reporter moiety: wherein the reporter moiety comprises a DNA, RNA or chimeric nucleic acid molecule that is operably linked to the blocked nucleic acid molecule and produces a detectable signal upon cleavage by RNP1 and/or RNP2; or wherein the reporter moiety comprises a DNA, RNA or chimeric nucleic acid molecule and is not operably linked to the blocked nucleic acid molecule and produces a detectable signal upon cleavage by RNP1 and/or RNP2. In some aspects, the detectable signal is produced within about 1-10 minutes upon binding of the target nucleic acid of interest to RNP1; in some aspects, the detectable signal is a fluorescent, chemiluminescent, radioactive, colorimetric or optical signal; and/or in some aspects, the reporter moiety comprises a modified nucleoside or nucleotide including but not limited to locked nucleic acids (LNAs), peptide nucleic acids (PNAs), 2′-O-methyl (2′-O-Me) modified nucleosides, 2′-fluoro (2′-F) modified nucleoside, and/or a phosphorothioate (PS) bonds.

In some aspects of the aforementioned embodiments, the template molecules do not comprise a complement of a PAM sequence, and in some aspects, the template molecules comprise a complement of a PAM sequence. In some aspects, the template molecules are single-stranded. In some aspects, the template molecules are linear; in yet other aspects the template molecules are circularized. In some aspects comprising circular blocked nucleic acid molecules, at least one of the plurality of circular high Kd blocked nucleic acid molecules comprises a first region comprising a sequence specific to the second guide RNA and a second region comprising a nuclease-cleavable sequence; where at least one circular high Kd blocked nucleic acid molecule comprises a nuclease-resistant DNA sequence in the first region and a nuclease-cleavable DNA sequence in the second region; at least one circular high Kd blocked nucleic acid molecule comprises a nuclease-resistant RNA sequence in the first region and a nuclease-cleavable DNA and RNA sequence in the second region; at least one circular high Kd blocked nucleic acid molecule comprises a nuclease-resistant DNA sequence in the first region and a nuclease-cleavable DNA and RNA sequence in the second region; or at least one circular high Kd blocked nucleic acid molecule comprises a nuclease-resistant RNA sequence in the first region and a nuclease-cleavable RNA sequence in the second region.

In some aspects the polymerase comprises strand displacement activity and/or 3′-to-5′ exonuclease activity, and in some aspects, the polymerase is Phi29 polymerase.

Yet another embodiment provides a composition of matter comprising a circular high Kd blocked nucleic acid molecule comprising: a region recognized by a gRNA in an RNP complex; a region comprising a sequence cleavable by a nucleic acid-guided nuclease in the RNP complex; and wherein the circular high Kd blocked nucleic acid molecule cannot activate the RNP complex, and wherein the circular high Kd blocked nucleic molecules are high Kd in relation to binding to the RNP complex.

A further embodiment provides a method of unblocking a circular high Kd blocked nucleic acid molecule comprising the steps of: (a) providing a circular high Kd blocked nucleic acid molecule comprising: a first region recognized by a gRNA in an RNP complex; a second region comprising a sequence cleavable by a nucleic acid-guided nuclease in the RNP complex, wherein the circular high Kd blocked nucleic acid molecule cannot substantially activate the RNP complex; and (b) initiating cleavage of the second region by the nucleic acid-guided nuclease in the RNP complex, wherein the circular high Kd blocked nucleic acid molecule becomes a linear low Kd unblocked nucleic acid molecule, and wherein the circular high Kd blocked nucleic acid molecules are high Kd and linear low K_(d) unblocked nucleic acid molecules are high K_(d) and low K_(d) in relation to binding the RNP complex.

Also provided as an embodiment is a cascade assay method of detecting a target nucleic acid of interest in a sample comprising the steps of: (a) providing a reaction mixture comprising: (i) a first ribonucleoprotein (RNP) complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA); wherein the first gRNA comprises a sequence complementary to a target nucleic acid of interest; (ii) a second ribonucleoprotein (RNP2) complex comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid molecule; and (iii) a plurality of circular blocked nucleic acid molecules comprising a sequence complementary to the second gRNA, wherein the circular blocked nucleic acid molecules cannot activate the RNP1 complex or the RNP2 complex; (b) contacting the reaction mixture with the sample under conditions that allow the target nucleic acid of interest in the sample to bind to RNP1; wherein upon binding of the target nucleic acid of interest, RNP1 becomes active initiating trans-cleavage of at least one of the circular blocked nucleic acid molecules thereby producing at least one linear unblocked nucleic acid molecule; the at least one linear unblocked nucleic acid molecule binds to RNP2 initiating cleavage of at least one further circular blocked nucleic acid molecule; and (c) detecting the cleavage products, thereby detecting the target nucleic acid of interest in the sample.

In some aspects, the RNP complex (either RNP1 or RNP2) comprises a nucleic acid-guided nuclease selected from Cas3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas14, Cas12h, Cas12i, Cas12j, Cas13a, or Cas13b, and in some aspects, the RNP complex comprises a nucleic acid-guided nuclease that is a Type V nucleic acid-guided nuclease or a Type VI nucleic acid-guided nuclease; the RNP complex comprises a nucleic acid-guided nuclease that exhibits both cis-cleavage and trans-cleavage activity; and/or the RNP complex comprises a nucleic acid-guided nuclease comprising a RuvC nuclease domain or a RuvC-like nuclease domain but lacks an HNH nuclease domain.

In some aspects of any embodiments comprising circular high K_(d) blocked nucleic acid molecules, the circular high K_(d) blocked nucleic acid molecule comprises a nuclease-resistant DNA sequence in the first region and a nuclease-cleavable DNA sequence in the second region; the circular high K_(d) blocked nucleic acid molecule comprises a nuclease-resistant RNA sequence in the first region and a nuclease-cleavable DNA and RNA sequence in the second region; the circular high K_(d) blocked nucleic acid molecule comprises a nuclease-resistant DNA sequence in the first region and a nuclease-cleavable DNA and RNA sequence in the second region; or the circular high K_(d) blocked nucleic acid molecule comprises a nuclease-resistant RNA sequence in the first region and a nuclease-cleavable RNA sequence in the second region.

In some aspects of these embodiments, the circular high K_(d) blocked nucleic acid molecule comprises 5′ and 3′ ends hybridized to each other and DNA, RNA, LNA or PNA bases having a high T_(m); and in some aspects, the K_(d) of the circular high K_(d) blocked nucleic acid molecules to the RNP complex or RNP2 is about 10⁵-fold greater, 10⁶-fold greater, 10⁷-fold greater, 10⁸-fold greater, 10⁹-fold greater, 10¹⁰-fold greater or more than the K_(d) of unblocked circular high K_(d) blocked nucleic acid molecules.

In some aspects the circular high K_(d) blocked nucleic acid molecule comprises a modified nucleoside or nucleotide, including but not limited to a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a 2′-O-methyl (2′-O-Me) modified nucleoside, a 2′-fluoro (2′-F) modified nucleoside, and/or a phosphorothioate (PS) bond.

In some aspects the circular high K_(d) blocked nucleic acid molecule is a single-stranded, double-stranded, or partially double-stranded molecule comprising one or more different combinations of DNA-DNA, DNA-RNA or RNA-RNA hybrid molecules. In some aspects the circular high K_(d) blocked nucleic acid molecule is a circular high K_(d) primer molecule. In some aspects the circular high K_(d) blocked nucleic acid molecule does not comprise a PAM sequence or the circular high K_(d) blocked nucleic acid molecule comprises a PAM sequence.

In some aspects of the aforementioned embodiments, the compositions of matter or reaction further comprises a reporter moiety wherein the reporter moiety comprises a DNA, RNA or chimeric nucleic acid molecule that is operably linked to the blocked nucleic acid molecule and produces a detectable signal upon cleavage by RNP1 and/or RNP2; or wherein the reporter moiety comprises a DNA, RNA or chimeric nucleic acid molecule and is not operably linked to the blocked nucleic acid molecule and produces a detectable signal upon cleavage by RNP1 and/or RNP2. In some aspects, the detectable signal is produced within about 1-10 minutes upon binding of the target nucleic acid of interest to RNP1; in some aspects, the detectable signal is a fluorescent, chemiluminescent, radioactive, colorimetric or optical signal; and/or in some aspects, the reporter moiety comprises a modified nucleoside or nucleotide including but not limited to locked nucleic acids (LNAs), peptide nucleic acids (PNAs), 2′-O-methyl (2′-O-Me) modified nucleosides, 2′-fluoro (2′-F) modified nucleoside, and/or a phosphorothioate (PS) bonds.

Yet another embodiment provides a composition of matter comprising: (a) a first preassembled ribonucleoprotein complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA that is specific to a target nucleic acid of interest, wherein the first nucleic acid-guided nuclease exhibits cis-cleavage activity and trans-cleavage activity; (b) a second preassembled ribonucleoprotein complex (RNP2) comprising a second nucleic acid-guided nuclease and a second guide RNA, wherein the second nucleic acid-guided nuclease exhibits cis-cleavage activity and trans-cleavage activity; and (c) a plurality of circular high K_(d) blocked nucleic acid molecules comprising a sequence complementary to the second gRNA, wherein the circular high K_(d) blocked nucleic acid molecules are not recognized by the RNP1 or RNP2, and wherein the circular high K_(d) blocked nucleic acid molecules are high K_(d) in relation to binding to RNP2.

Another embodiment provides a composition of matter comprising: (a) a first preassembled ribonucleoprotein complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA that is specific to a target nucleic acid of interest, wherein the first nucleic acid-guided nuclease exhibits cis-cleavage activity and trans-cleavage activity; (b) a second preassembled ribonucleoprotein complex (RNP2) comprising a second nucleic acid-guided nuclease and a second guide RNA, wherein the second nucleic acid-guided nuclease exhibits cis-cleavage activity and trans-cleavage activity; and (c) a plurality of engineered linear high K_(d) blocked nucleic acid molecules comprising a first sequence complementary to the second gRNA, wherein the linear high K_(d) blocked nucleic acid molecules are not recognized by the RNP1 and RNP2, and wherein the linear high K_(d) blocked nucleic acid molecules are high K_(d) in relation to binding to the RNP2.

Yet another embodiment provides a composition of matter comprising: (a) a first preassembled ribonucleoprotein complex (RNP1) comprising a first nucleic acid-guided nuclease and a first guide RNA that is specific to a target nucleic acid of interest, wherein the first nucleic acid-guided nuclease exhibits cis-cleavage activity and trans-cleavage activity; (b) a second preassembled ribonucleoprotein complex (RNP2) comprising a second nucleic acid-guided nuclease and a second guide RNA, wherein the second nucleic acid-guided nuclease exhibits cis-cleavage activity and trans-cleavage activity; and (c) a plurality of engineered high K_(d) blocked nucleic acid molecules comprising a sequence complementary to the second gRNA, wherein the high K_(d) blocked nucleic acid molecules are not recognized by the RNP1 and RNP2, and wherein the high K_(d) blocked nucleic acid molecules are high K_(d) in relation to binding to the RNP complex.

In aspects of any one of the foregoing embodiments, the high K_(d) blocked nucleic acid molecule comprises DNA, RNA, LNA or PNA bases having a high Tm; the 5′ and 3′ ends of the high K_(d) blocked nucleic acid molecule comprise phosphorothioate bonds (PS); high K_(d) blocked nucleic acid molecule comprises one or more different combinations of DNA-DNA, DNA-RNA or RNA-RNA hybrid molecules; and/or the high K_(d) blocked nucleic acid molecule comprises a nucleic acid region comprising nanoparticles attached thereto, wherein the nanoparticles provide steric hindrance to internalization in RNP2 and block RNP2 activation until cleavage and removal of the nucleic acid region comprising the nanoparticles.

In other aspects, the first and/or second nucleic acid-guided nuclease is a Cas3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas14, Cas12h, Cas12i, Cas12j, Cas13a, Cas13b nuclease; in some aspects, the first nucleic acid-guided nuclease can is a different nucleic acid-guided nuclease than the second nucleic acid-guided nuclease; in some aspects, the first and/or second nucleic acid-guided nuclease is a Type V nucleic acid-guided nuclease or a Type VI nucleic acid-guided nuclease and/or in some aspects, the first and/or second nucleic acid-guided nuclease comprises a RuvC nuclease domain or a RuvC-like nuclease domain and lacks an HNH nuclease domain.

Aspects also include the composition of matter comprises about 1 fM to about 10 μM of the RNP1; and/or the composition of matter comprises about 1 fM to about 1 mM of the RNP2.

In some aspects the composition of matter comprises at least two different RNP1 complex species, wherein different RNP1s comprise different gRNA sequences; and in some aspects the composition comprises 2 to 10000 different RNP1s, 2 to 1000 different RNP1s, 2 to 100 different RNP1s, or 2 to 10 different RNP1s.

In some aspects the RNP2 recognizes a PAM sequence, and in other aspects the RNP2 complex does not recognize a PAM sequence.

In some aspects of the aforementioned embodiments, the composition of matter further comprises a reporter moiety, wherein the reporter moiety comprises a DNA, RNA or chimeric nucleic acid molecule that is operably linked to the blocked nucleic acid molecule and produces a detectable signal upon cleavage by RNP1 and/or RNP2; or wherein the reporter moiety comprises a DNA, RNA or chimeric nucleic acid molecule and is not operably linked to the blocked nucleic acid molecule and produces a detectable signal upon cleavage by RNP1 and/or RNP2. In some aspects, the detectable signal is produced within about 1-10 minutes upon binding of the target nucleic acid of interest to RNP1; in some aspects, the detectable signal is a fluorescent, chemiluminescent, radioactive, colorimetric or optical signal; and/or in some aspects, the reporter moiety comprises a modified nucleoside or nucleotide including but not limited to locked nucleic acids (LNAs), peptide nucleic acids (PNAs), 2′-O-methyl (2′-O-Me) modified nucleosides, 2′-fluoro (2′-F) modified nucleoside, and/or a phosphorothioate (PS) bonds.

In some aspects the high Kd blocked nucleic acid molecule is a high Kd blocked primer molecule.

In some aspects the linear high K_(d) blocked nucleic acid molecule is converted to a linear low K_(d) blocked nucleic acid molecule upon trans-cleavage by RNP1 and/or RNP2. In some aspects the K_(d) of the blocked nucleic acid molecules to the RNP2 is about 10⁵-fold greater, 10⁶-fold greater, 10⁷-fold greater, 10⁸-fold greater, 10⁹-fold greater, 10¹⁰-fold greater or more than the K_(d) of unblocked nucleic acid molecules.

In some aspects of the compositions of matter comprising circular blocked nucleic acid molecules, at least one of the plurality of circular high Kd blocked nucleic acid molecules comprises a first region comprising a sequence specific to the second guide RNA and a second region comprising a nuclease-cleavable sequence; where at least one circular high Kd blocked nucleic acid molecule comprises a nuclease-resistant DNA sequence in the first region and a nuclease-cleavable DNA sequence in the second region; at least one circular high Kd blocked nucleic acid molecule comprises a nuclease-resistant RNA sequence in the first region and a nuclease-cleavable DNA and RNA sequence in the second region; at least one circular high Kd blocked nucleic acid molecule comprises a nuclease-resistant DNA sequence in the first region and a nuclease-cleavable DNA and RNA sequence in the second region; or at least one circular high Kd blocked nucleic acid molecule comprises a nuclease-resistant RNA sequence in the first region and a nuclease-cleavable RNA sequence in the second region.

In some aspects of the compositions of matter comprising linear blocked nucleic acid molecules, the linear high K_(d) nucleic acid molecules comprise a structure represented by any one of Formulas I-IV, where Formulas I-IV comprise in the 5′-to-3′ direction:

(a)A-(B-L)_(J)-C-M-T-D  (Formula I);

-   -   wherein A is 0-15 nucleotides in length;     -   B is 4-12 nucleotides in length;     -   L is 3-25 nucleotides in length;     -   J is an integer between 1 and 10;     -   C is 4-15 nucleotides in length;     -   M is 1-25 nucleotides in length or is absent, wherein if M is         absent then A-(B-L)_(J)-C and T-D are separate nucleic acid         strands;     -   T is 17-135 nucleotides in length and comprises at least 50%         sequence complementarity to B and C; and     -   D is 0-10 nucleotides in length and comprises at least 50%         sequence complementarity to A;

(b)D-T-T′-C-(L-B)_(J)-A  (Formula II);

-   -   wherein D is 0-10 nucleotides in length;     -   T-T′ is 17-135 nucleotides in length;     -   T′ is 1-10 nucleotides in length and does not hybridize with T;     -   C is 4-15 nucleotides in length and comprises at least 50%         sequence complementarity to T;     -   L is 3-25 nucleotides in length and does not hybridize with T;     -   B is 4-12 nucleotides in length and comprises at least 50%         sequence complementarity to T;     -   J is an integer between 1 and 10;     -   A is 0-15 nucleotides in length and comprises at least 50%         sequence complementarity to D;

(c)T-D-M-A-(B-L)_(J)-C  (Formula III);

-   -   wherein T is 17-135 nucleotides in length;     -   D is 0-10 nucleotides in length;     -   M is 1-25 nucleotides in length or is absent, wherein if M is         absent then T-D and A-(B-L)_(J)-C are separate nucleic acid         strands;     -   A is 0-15 nucleotides in length and comprises at least 50%         sequence complementarity to D;     -   B is 4-12 nucleotides in length and comprises at least 50%         sequence complementarity to T;     -   L is 3-25 nucleotides in length;     -   J is an integer between 1 and 10; and     -   C is 4-15 nucleotides in length; or

(d)T-D-M-A-L_(p)-C  (Formula IV);

-   -   wherein T is 17-31 nucleotides in length (e.g., 17-100, 17-50,         or 17-25);     -   D is 0-15 nucleotides in length;     -   M is 1-25 nucleotides in length;     -   A is 0-15 nucleotides in length and comprises a sequence         complementary to D; and     -   L is 3-25 nucleotides in length;     -   p is 0 or 1;     -   C is 4-15 nucleotides in length and comprises a sequence         complementary to T.

And in Some Aspects,

-   -   (a) T of Formula I comprises at least 80% sequence         complementarity to B and C;     -   (b) D of Formula I comprises at least 80% sequence         complementarity to A;     -   (c) C of Formula II comprises at least 80% sequence         complementarity to T;     -   (d) B of Formula II comprises at least 80% sequence         complementarity to T;     -   (e) A of Formula II comprises at least 80% sequence         complementarity to D;     -   (f) A of Formula III comprises at least 80% sequence         complementarity to D;     -   (g) B of Formula III comprises at least 80% sequence         complementarity to T;     -   (h) A of Formula IV comprises at least 80% sequence         complementarity to D; and/or     -   (i) C of Formula IV comprises at least 80% sequence         complementarity to T.

In some aspects, at least one of the linear blocked nucleic acid molecules include the sequence of any one of SEQ ID NOs: 14-1421.

In another embodiment, there is provided a method for syndromic testing comprising: (a) providing a reaction mixture comprising: (i) a plurality of first ribonucleoprotein complexes (RNP1s), each RNP1 comprising a nucleic acid-guided nuclease exhibiting both cis-cleavage and trans-cleavage activity and a first guide RNA (gRNA), wherein the first gRNA comprises a sequence complementary to a target nucleic acid of interest, and wherein the reaction mixture comprises at least two different RNP1s, wherein different RNP1s comprise different first gRNAs; (ii) a second ribonucleoprotein complex (RNP2), wherein the RNP2 comprises a second nucleic acid-guided nuclease and a second gRNA that does not recognize any of the target nucleic acids of interest; and (iii) a plurality of blocked nucleic acid molecules comprising a sequence complementary to the second gRNA, wherein the blocked nucleic acid molecule cannot substantially activate the plurality of RNP1s or the RNP2; (b) contacting the reaction mixture with a sample under conditions that allow target nucleic acids of interest in the sample to bind to the RNP1s, wherein: upon binding of any one of the target nucleic acids of interest, the RNP1 becomes active, cleaving at least one of the blocked nucleic acid molecules, thereby producing at least one unblocked nucleic acid molecule; and at least one unblocked nucleic acid molecule binds to the second gRNA thereby activating RNP2 and initiating trans-cleavage of at least one further blocked nucleic acid molecule; and (c) detecting products of the cleavage of step (b), thus identifying at least one target nucleic acid of interest in the sample.

In some aspects of this embodiment, the first and/or second nucleic acid-guided nuclease is a Cas3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas14, Cas12h, Cas12i, Cas12j, Cas13a, Cas13b nuclease; in some aspects, the first nucleic acid-guided nuclease can is a different nucleic acid-guided nuclease than the second nucleic acid-guided nuclease; in some aspects, the first and/or second nucleic acid-guided nuclease is a Type V nucleic acid-guided nuclease or a Type VI nucleic acid-guided nuclease and/or in some aspects, the first and/or second nucleic acid-guided nuclease comprises a RuvC nuclease domain or a RuvC-like nuclease domain and lacks an HNH nuclease domain.

Aspects also include the reaction mixture comprises about 1 fM to about 10 μM of the RNP1; and/or the reaction mixture comprises about 1 fM to about 1 mM of the RNP2. In some aspects the reaction mixture comprises at least two different RNP1 complex species, wherein different RNP1s comprise different gRNA sequences; and in some aspects the composition comprises 2 to 10000 different RNP1s, 2 to 1000 different RNP1s, 2 to 100 different RNP1s, or 2 to 10 different RNP1s.

In some aspects the K_(d) of the plurality of blocked nucleic acid molecules to the RNP2 is about 10⁵-fold greater, 10⁶-fold greater, 10⁷-fold greater, 10⁸-fold greater, 10⁹-fold greater, 10¹⁰-fold greater or more than the K_(d) of unblocked nucleic acid molecules.

In some aspects of the aforementioned embodiment, the target nucleic acid of interest is of bacterial, viral, fungal, or mammalian origin, and in some aspects, the sample may include peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, and/or umbilical cord blood.

In some aspects of the aforementioned embodiments, the reaction mixture further comprises a reporter moiety: wherein the reporter moiety comprises a DNA, RNA or chimeric nucleic acid molecule that is operably linked to the blocked nucleic acid molecule and produces a detectable signal upon cleavage by RNP1 and/or RNP2; or wherein the reporter moiety comprises a DNA, RNA or chimeric nucleic acid molecule and is not operably linked to the blocked nucleic acid molecule and produces a detectable signal upon cleavage by RNP1 and/or RNP2. In some aspects, the detectable signal is produced within about 1-10 minutes upon binding of the target nucleic acid of interest to RNP1; in some aspects, the detectable signal is a fluorescent, chemiluminescent, radioactive, colorimetric or optical signal; and/or in some aspects, the reporter moiety comprises a modified nucleoside or nucleotide including but not limited to locked nucleic acids (LNAs), peptide nucleic acids (PNAs), 2′-O-methyl (2′-O-Me) modified nucleosides, 2′-fluoro (2′-F) modified nucleoside, and/or a phosphorothioate (PS) bonds. In some aspects the detectable signal is produced within about 1-10 minutes upon the target nucleic acid of interest activating RNP1.

In some aspects the blocked nucleic acid molecules comprise a PAM sequence and in other aspects, the blocked nucleic acid molecules do not comprise a PAM sequence. In some aspects the blocked nucleic acid molecules are linear and in some aspects, the blocked nucleic acids are circular and in yet other aspects, the blocked nucleic acid molecules are a mixture of circular and linear blocked nucleic acid molecules.

In some aspects the blocked nucleic acid molecules are blocked primer molecules and wherein the reaction mixture further comprises a polymerase and nucleotides.

In some aspects, the syndromic testing is for any two or more of common flu (e.g., influenza A, influenza A/H1, influenza A/H3, influenza A/H1-2009, and influenza B); one of the multiple strains of respiratory syncytial virus (RSV), such as RSV-A and RSV-B; at least one variant of SARS-CoV-2 (e.g., B.1.1.7, B.1.351, P.1, B.1.617.2, BA.1, BA.2, BA.2.12.1, BA.4, and BA.5); and at least one of other pathogens of interest (e.g., parainfluenza virus 1-4, human metapneumovirus, human rhinovirus, human enterovirus, adenovirus, coronavirus HKU1, coronavirus NL63, coronavirus 229E, coronavirus OC43, MERS).

Yet other embodiments provide: a method of detecting a target nucleic acid of interest in a sample comprising the steps of: providing a reaction mixture comprising a first RNP complex comprising a first nucleic acid-guided nuclease and a first guide RNA (gRNA), wherein the first gRNA comprises a sequence complementary to a target nucleic acid of interest; and a second RNP complex comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest; and contacting the reaction mixture with the sample under conditions that allow the target nucleic acid of interest in the sample to bind to the first gRNA, wherein upon binding of the target nucleic acid of interest, the first RNP complex becomes active which catalyzes activation of the second RNP complex via one or more blocked nucleic acids to produce a detectable signal from a reporter moiety.

A further embodiment provides a modular cascade assay comprising: a first nucleic acid-guided nuclease, wherein the first nucleic acid-guided nuclease will form a first ribonucleoprotein complex with a first gRNA that is complementary to a target nucleic acid of interest; a second RNP2 complex comprising a second nucleic acid-guided nuclease and a second gRNA that is not complementary to a target nucleic acid of interest; and a plurality of blocked nucleic acid molecules comprising a sequence complementary to the second gRNA, wherein the blocked nucleic acid molecules cannot activate the RNP1 complex or the RNP2 complex; wherein by changing the sequence of the first gRNA, the modular cascade assay is changed to detect different target nucleic acids of interest.

These aspects and other features and advantages of the invention are described below in more detail.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings in which:

FIG. 1A is an overview of a prior art assay where target nucleic acids of interest from a sample must be amplified before performing a detection assay.

FIG. 1B is an overview of the general principles underlying the nucleic acid-guided nuclease cascade assay described in detail herein where target nucleic acids of interest from a sample do not need to be amplified before detection.

FIG. 2A is a diagram showing the sequence of steps in an exemplary cascade assay utilizing blocked nucleic acids.

FIG. 2B is a diagram showing an exemplary blocked nucleic acid molecule and a method for unblocking the blocked nucleic acid molecules of the disclosure.

FIG. 2C shows schematics of several exemplary blocked nucleic acid molecules containing the structure of Formula I, as described herein.

FIG. 2D shows schematics of several exemplary blocked nucleic acid molecules containing the structure of Formula II, as described herein.

FIG. 2E shows schematics of several exemplary blocked nucleic acid molecules containing the structure of Formula III, as described herein.

FIG. 2F shows schematics of several exemplary blocked nucleic acid molecules containing the structure of Formula IV, as described herein.

FIG. 2G shows an exemplary single-stranded blocked nucleic acid molecule with a design able to block R-loop formation with an RNP complex, thereby blocking activation of the trans-nuclease activity of an RNP complex (i.e., RNP2).

FIG. 2H shows schematics of exemplary circularized blocked nucleic acid molecules.

FIG. 3A is a diagram showing the sequence of steps in an exemplary cascade assay involving circular blocked primer molecules and linear template molecules.

FIG. 3B is a diagram showing the sequence of steps in an exemplary cascade assay involving circular blocked primer molecules and circular template molecules.

FIG. 4 illustrates three embodiments of reporter moieties.

FIG. 5A shows a lateral flow assay that can be used to detect the cleavage and separation of a signal from a reporter moiety.

FIG. 5B shows a schematic of a lateral flow assay device illustrating the results of an exemplary syndromic test.

FIG. 6 shows a titered quantification of a synthesized nucleocapsid gene (N-gene) using the nucleic acid detection methods described herein. As described in Example VI, a cascade assay was initiated using the detection methods described in Examples II-V above.

FIG. 7 shows titered quantification of an inactivated SARS-CoV-2 virus using the nucleic acid detection methods described in Examples II-V above.

FIG. 8 shows titered quantification of DNA from Methicillin-resistant Staphylococcus (MRSA) using the nucleic acid detection methods described in Examples II-V.

FIG. 9 shows titered quantification of DNA from Methicillin-resistant Staphylococcus (MRSA) using the nucleic acid detection methods described in Examples II-V.

FIG. 10 shows the detection of 3 copies of a molecule of DNA from Methicillin-resistant Staphylococcus (MRSA) using Molecule C5 as the blocked nucleic acid molecule.

FIG. 11 shows the detection of 3 copies of a molecule of DNA from Methicillin-resistant Staphylococcus (MRSA) using Molecule C6 as the blocked nucleic acid molecule.

FIG. 12 shows the detection of 3 copies of a molecule of DNA from Methicillin-resistant Staphylococcus (MRSA) using Molecule C7 as the blocked nucleic acid molecule.

FIG. 13 shows the detection of 3 copies of a molecule of DNA from Methicillin-resistant Staphylococcus (MRSA) using Molecule C8 as the blocked nucleic acid molecule.

FIG. 14 shows the detection of 3 copies of a molecule of DNA from Methicillin-resistant Staphylococcus (MRSA) using Molecule C9 as the blocked nucleic acid molecule.

It should be understood that the drawings are not necessarily to scale, and that like reference numbers refer to like features.

Definitions

All of the functionalities described in connection with one embodiment of the compositions and methods described herein are intended to be applicable to the additional embodiments of the compositions and methods described herein except where expressly stated or where the feature or function is incompatible with the additional embodiments. For example, where a given feature or function is expressly described in connection with one embodiment but not expressly mentioned in connection with an alternative embodiment, it should be understood that the feature or function may be deployed, utilized, or implemented in connection with the alternative embodiment unless the feature or function is incompatible with the alternative embodiment.

Note that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” refers to one or more cells, and reference to “a system” includes reference to equivalent steps, methods and devices known to those skilled in the art, and so forth. Additionally, it is to be understood that terms such as “left,” “right,” “top,” “bottom,” “front,” “rear,” “side,” “height,” “length,” “width,” “upper,” “lower,” “interior,” “exterior,” “inner,” “outer” that may be used herein merely describe points of reference and do not necessarily limit embodiments of the present disclosure to any particular orientation or configuration. Furthermore, terms such as “first,” “second,” “third,” etc., merely identify one of a number of portions, components, steps, operations, functions, and/or points of reference as disclosed herein, and likewise do not necessarily limit embodiments of the present disclosure to any particular configuration or orientation.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing devices, formulations and methodologies that may be used in connection with the presently described invention. Conventional methods are used for the procedures described herein, such as those provided in the art, and demonstrated in the Examples and various general references. Unless otherwise stated, nucleic acid sequences described herein are given, when read from left to right, in the 5′ to 3′ direction. Nucleic acid sequences may be provided as DNA, as RNA, or a combination of DNA and RNA (e.g., a chimeric nucleic acid).

Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

The term “and/or” where used herein is to be taken as specific disclosure of each of the multiple specified features or components with or without another. Thus, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention. The terms used herein are intended to have the plain and ordinary meaning as understood by those of ordinary skill in the art.

As used herein, the term “about,” as applied to one or more values of interest, refers to a value that falls within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of a stated reference value, unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

As used herein, the terms “binding affinity” or “dissociation constant” or “K_(d)” refer to the tendency of a molecule to bind (covalently or non-covalently) to a different molecule. A high K_(d) (which in the context of the present disclosure refers to blocked nucleic acid molecules or blocked primer molecules binding to RNP2) indicates the presence of more unbound molecules, and a low K_(d) (which in the context of the present disclosure refers to unblocked nucleic acid molecules or unblocked primer molecules binding to RNP2) indicates the presence of more bound molecules. In the context of the present disclosure and the binding of blocked or unblocked nucleic acid molecules or blocked or unblocked primer molecules to RNP2, aow K_(d) values are in a range from about 100 fM to about 1 aM or lower (e.g., 100 zM) and high K_(d) values are in the range of 100 nM-100 μM (10 mM) and thus are about 10⁵- to 10¹⁰-fold or higher as compared to low K_(d) values.

As used herein, the terms “binding domain” or “binding site” refer to a region on a protein, DNA, or RNA, to which specific molecules and/or ions (ligands) may form a covalent or non-covalent bond. By way of example, a polynucleotide sequence present on a nucleic acid molecule (e.g., a primer binding domain) may serve as a binding domain for a different nucleic acid molecule (e.g., an unblocked primer nucleic acid molecule). Characteristics of binding sites are chemical specificity, a measure of the types of ligands that will bond, and affinity, which is a measure of the strength of the chemical bond.

As used herein, the term “blocked nucleic acid molecule” refers to nucleic acid molecules that cannot bind to the first or second RNP complex to activate cis- or trans-cleavage. “Unblocked nucleic acid molecule” refers to a formerly blocked nucleic acid molecule that can bind to the second RNP complex (RNP2) to activate trans-cleavage of additional blocked nucleic acid molecules.

The terms “Cas RNA-guided endonuclease” or “CRISPR nuclease” or “nucleic acid-guided nuclease” refer to a CRISPR-associated protein that is an RNA-guided endonuclease suitable for assembly with a sequence-specific gRNA to form a ribonucleoprotein (RNP) complex.

As used herein, the terms “cis-cleavage”, “cis-endonuclease activity”, “cis-mediated endonuclease activity”, “cis-nuclease activity”, “cis-mediated nuclease activity”, and variations thereof refer to sequence-specific cleavage of a target nucleic acid of interest, including an unblocked nucleic acid molecule or synthesized activating molecule, by a nucleic acid-guided nuclease in an RNP complex. Cis-cleavage is a single turn-over cleavage event in that only one substrate molecule is cleaved per event.

The term “complementary” as used herein refers to Watson-Crick base pairing between nucleotides and specifically refers to nucleotides hydrogen-bonded to one another with thymine or uracil residues linked to adenine residues by two hydrogen bonds and cytosine and guanine residues linked by three hydrogen bonds. In general, a nucleic acid includes a nucleotide sequence described as having a “percent complementarity” or “percent homology” to a specified second nucleotide sequence. For example, a nucleotide sequence may have 80%, 90%, or 100% complementarity to a specified second nucleotide sequence, indicating that 8 of 10, 9 of 10, or 10 of 10 nucleotides of a sequence are complementary to the specified second nucleotide sequence. For instance, the nucleotide sequence 3′-TCGA-5′ is 100% complementary to the nucleotide sequence 5′-AGCT-3′; and the nucleotide sequence 3′-TCGATCGATCGA-5′ [SEQ ID NO: 1] is 100% complementary to a region of the nucleotide sequence 5′-GCTAGCTAGC-3′ [SEQ ID NO: 2].

As used herein, the term “contacting” refers to placement of two moieties in direct physical association, including in solid or liquid form. Contacting can occur in vitro with isolated cells (for example in a tissue culture dish or other vessel) or in vivo by administering an agent to a subject.

A “control” is a reference standard of a known value or range of values.

The terms “guide nucleic acid” or “guide RNA” or “gRNA” refer to a polynucleotide comprising 1) a crRNA region or guide sequence capable of hybridizing to the target strand of a target nucleic acid of interest, and 2) a scaffold sequence capable of interacting or complexing with a nucleic acid-guided nuclease. The crRNA region of the gRNA is a customizable component that enables specificity in every nucleic acid-guided nuclease reaction. A gRNA can include any polynucleotide sequence having sufficient complementarity with a target nucleic acid of interest to hybridize with the target nucleic acid of interest and to direct sequence-specific binding of a ribonucleoprotein (RNP) complex containing the gRNA and nucleic acid-guided nuclease to the target nucleic acid. Target nucleic acids of interest may include a protospacer adjacent motif (PAM), and, following gRNA binding, the nucleic acid-guided nuclease induces a double-stranded break either inside or outside the protospacer region on the target nucleic acid of interest, including on an unblocked nucleic acid molecule or synthesized activating molecule. A gRNA may contain a spacer sequence including a plurality of bases complementary to a protospacer sequence in the target nucleic acid. For example, a spacer can contain about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more bases. The gRNA spacer may be 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or more complementary to its corresponding target nucleic acid of interest. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences. A guide RNA may be from about 20 nucleotides to about 300 nucleotides long. Guide RNAs may be produced synthetically or generated from a DNA template.

“Modified” refers to a changed state or structure of a molecule. Molecules may be modified in many ways including chemically, structurally, and functionally. In one embodiment, a nucleic acid molecule (for example, a blocked nucleic acid molecule) may be modified by the introduction of non-natural nucleosides, nucleotides, and/or internucleoside linkages. In another embodiment, a modified protein (e.g., a nucleic acid-guided nuclease) may refer to any polypeptide sequence alteration which is different from the wildtype.

The terms “percent sequence identity”, “percent identity”, or “sequence identity” refer to percent (%) sequence identity with respect to a reference polynucleotide or polypeptide sequence following alignment by standard techniques. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, PSI-BLAST, or Megalign software. In some embodiments, the software is MUSCLE (Edgar, Nucleic Acids Res., 32(5):1792-1797 (2004)). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, in embodiments, percent sequence identity values are generated using the sequence comparison computer program BLAST (Altschul et al., J. Mol. Biol., 215:403-410 (1990)).

As used herein, the terms “preassembled ribonucleoprotein complex”, “ribonucleoprotein complex”, “RNP complex”, or “RNP” refer to a complex containing a guide RNA (gRNA) and a nucleic acid-guided nuclease, where the gRNA is integrated with the nucleic acid-guided nuclease. The gRNA, which includes a sequence complementary to a target nucleic acid of interest, guides the RNP to the target nucleic acid of interest and hybridizes to it. The hybridized target nucleic acid-gRNA units are cleaved by the nucleic acid-guided nuclease. In the cascade assays described herein, a first ribonucleoprotein complex (RNP1) includes a first guide RNA (gRNA) specific to a nucleic acid target nucleic acid of interest, and a first nucleic acid-guided nuclease, such as, for example, cas12a or cas14a for a DNA target nucleic acid, or cas13a for an RNA target nucleic acid. A second ribonucleoprotein complex (RNP2) for signal amplification includes a second guide RNA specific to an unblocked nucleic acid or synthesized activating molecule, and a second nucleic acid-guided nuclease, which may be different from or the same as the first nucleic acid-guided nuclease.

As used herein, the terms “protein” and “polypeptide” are used interchangeably. Proteins may or may not be made up entirely of amino acids.

As used herein, the term “sample” refers to tissues; cells or component parts; body fluids, including but not limited to peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, and umbilical cord blood; food; agricultural products; pharmaceuticals; cosmetics, nutriceuticals; personal care products; environmental substances such as soil, water, or air; industrial sites and products; and chemicals and compounds. A sample further may include a homogenate, lysate or extract. A sample further refers to a medium, such as a nutrient broth or gel, which may contain cellular components, such as proteins or nucleic acid molecules.

The terms “target DNA sequence”, “target sequence”, “target nucleic acid of interest”, “target molecule of interest”, “target nucleic acid”, or “target of interest” refer to any locus that is recognized by a gRNA sequence in vitro or in vivo. The “target strand” of a target nucleic acid of interest is the strand of the double-stranded target nucleic acid that is complementary to a gRNA. The spacer sequence of a gRNA may be 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 98%, 99% or more complementary to the target nucleic acid of interest. Optimal alignment can be determined with the use of any suitable algorithm for aligning sequences. Full complementarity is not necessarily required provided there is sufficient complementarity to cause hybridization and trans-cleavage activation of an RNP complex. A target nucleic acid of interest can include any polynucleotide, such as DNA (ssDNA or dsDNA) or RNA polynucleotides. A target nucleic acid of interest may be located in the nucleus or cytoplasm of a cell such as, for example, within an organelle of a eukaryotic cell, such as a mitochondrion or a chloroplast, or it can be exogenous to a host cell, such as a eukaryotic cell or a prokaryotic cell. The target nucleic acid of interest may be present in a sample, such as a biological or environmental sample, and it can be a viral nucleic acid molecule, a bacterial nucleic acid molecule, a fungal nucleic acid molecule, or a polynucleotide of another organism, such as a coding or a non-coding sequence, and it may include single-stranded or double-stranded DNA molecules, such as a cDNA or genomic DNA, or RNA molecules, such as mRNA, tRNA, and rRNA. The target nucleic acid may be associated with a protospacer adjacent motif (PAM) sequence, which may include a 2-5 base pair sequence adjacent to the protospacer. In some embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more target nucleic acids can be detected by the disclosed method.

As used herein, the terms “trans-cleavage”, “trans-endonuclease activity”, “trans-mediated endonuclease activity”, “trans-nuclease activity”, “trans-mediated nuclease activity”, and variations thereof, refer to indiscriminate, non-sequence-specific cleavage of a nucleic acid molecule by an endonuclease (such as by a Cas12, Cas13, and Cas14) which is triggered by cis-(sequence-specific) cleavage. Trans-cleavage is a “multiple turn-over” event, in that more than one substrate molecule is cleaved after initiation by a single turn-over cis-cleavage event.

Type V CRISPR/Cas nucleic acid-guided nucleases are a subtype of Class 2 CRISPR/Cas effector nucleases such as, but not limited to, engineered Cas12a, Cas12b, Cas12c, C2c4, C2c8, C2c5, C2c10, C2c9, CasX (Cas12e), CasY (Cas12d), Cas 13a nucleases or naturally-occurring proteins, such as a Cas12a isolated from, for example, Francisella tularensis subsp. novicida (Gene ID: 60806594), Candidatus Methanoplasma termitum (Gene ID: 24818655), Candidatus Methanomethylophilus alvus (Gene ID: 15139718), and [Eubacterium] eligens ATCC 27750 (Gene ID: 41356122), and an artificial polypeptide, such as a chimeric protein.

The term “variant” refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many if not most regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions). A variant of a polypeptide may be a conservatively modified variant. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code (e.g., a non-natural amino acid). A variant of a polypeptide may be naturally occurring, such as an allelic variant, or it may be a variant that is not known to occur naturally. Variants include modifications-including chemical modifications—to one or more amino acids that do not involve amino acid substitutions, additions or deletions.

A “vector” is any of a variety of nucleic acids that comprise a desired sequence or sequences to be delivered to and/or expressed in a cell. Vectors are typically composed of DNA, although RNA vectors are also available. Vectors include, but are not limited to, plasmids, fosmids, phagemids, virus genomes, synthetic chromosomes, and the like.

DETAILED DESCRIPTION

The present disclosure provides compositions of matter, methods, and cascade assays for detecting nucleic acids. The cascade assays described herein comprise first and second ribonucleoprotein complexes and either blocked nucleic acid molecules or blocked primer molecules. The blocked nucleic acid molecules or blocked primer molecules keep the second ribonucleoprotein complexes “locked” unless and until a target nucleic acid of interest activates the first ribonucleoprotein complex. The methods comprise the steps of providing cascade assay components, contacting the cascade assay components with a sample, and detecting a signal that is generated only when a target nucleic acid of interest is present in the sample ids.

Early and accurate detection and determination of infections and diseases is crucial for appropriate prevention strategies, accurate testing, confirmation, and further diagnosis and treatment. Nucleic acid-guided nucleases, such as the Cas12a endonuclease, can be utilized as diagnostic tools for the detection of target nucleic acids of interest associated with diseases. However, currently available state-of-the-art CRISPR Cas12a-based nucleic acid detection relies on DNA amplification before using Cas12a enzymes, which significantly hinders the ability to perform rapid point-of-care testing. The lack of rapidity is due to the fact that target-specific activation of Cas12a enzymes, referred herein as cis-cleavage, is a single turnover event in which the number of activated enzyme complexes is, at most, equal to the number of copies of the target nucleic acids of interest in the sample. Once a ribonucleoprotein (RNP) complex is activated after completion of cis-cleavage, the RNP complex initiates rapid non-specific trans-endonuclease activity, which is a multi-turnover event. Some currently available methods use trans-cleavage to cleave fluorescent reporters that are initially quenched to generate a signal, thereby indicating the presence of a cis-cleavage event—the target nucleic acid. However, the K_(cat) of activated Cas12a complex is 17/sec and 3/sec for dsDNA and ssDNA targets, respectively. Therefore, for less than 10,000 target copies, the number of reporters cleaved is not sufficient to generate a signal in less than 60 minutes. Hence, all current technologies rely on DNA amplification to first generate billions of target copies to activate a proportional number of ribonucleoprotein complexes to generate a detectable signal in 30-60 minutes.

The present disclosure describes a nucleic acid-guided nuclease cascade assay that can detect one or more target nucleic acids of interest (e.g., DNA, RNA and/or cDNA) at attamolar (aM) (or lower) limits in about 10 minutes or less without the need for amplifying the target nucleic acid(s) of interest, thereby avoiding the drawbacks of multiplex amplification, such as primer-dimerization. As described in detail below, the nucleic acid-guided nuclease cascade assays utilize signal amplification mechanisms comprising various components including nucleic acid-guided nucleases, guide RNAs (gRNAs), blocked nucleic acid molecules or blocked primer molecules, reporter moieties, and, in some embodiments, polymerases. A particularly advantageous feature of the cascade assay is that, with the exception of the gRNA (gRNA1) in RNP1, the cascade assay components stay the same no matter what target nucleic acid(s) of interest are being detected. In this sense, the cascade assay is modular.

FIG. 1A provides a simplified diagram demonstrating a prior art method (1) of a nucleic acid-guided nuclease detection assay where target nucleic acids of interest from a sample must be amplified in order to be detected. First, assuming the presence of a target nucleic acid of interest in a sample, the target nucleic acid of interest (2) is amplified to produce many copies of the target nucleic acid of interest (4). The detection assay is initiated (step 2) when the target nucleic acid of interest (4) is combined with and binds to a pre-assembled ribonucleoprotein complex (6), which is part of a reaction mix. The ribonucleoprotein complex (6) comprises a guide RNA (gRNA) and a nucleic acid-guided nuclease, where the gRNA is integrated with the nucleic acid-guided nuclease. The gRNA, which includes a sequence complementary to the target nucleic acid of interest, guides the RNP complex to the target nucleic acid of interest and hybridizes to it thereby activating the ribonucleoprotein complex (6). The nucleic acid-guided nuclease exhibits (i.e., possesses) both cis- and trans-cleavage activity, where trans-cleavage activity is initiated by cis-cleavage activity. Cis-cleavage activity occurs as the target nucleic acid of interest binds to the gRNA and is cleaved by the nucleic acid guided nuclease (i.e., activation). Once cis-cleavage of the target nucleic acid of interest is initiated, trans-cleavage activity is triggered, where trans-cleavage activity is indiscriminate, non-sequence-specific cleavage of nucleic acid molecules in the sample and is a multi-turnover event.

In step 3, the trans-cleavage activity triggers activation of reporter moieties (12) that are present in the reaction mix. The reporter moieties (12) may be a synthetic molecule linked or conjugated to a quencher (14) and a fluorophore (16) such as, for example, a probe with a dye label (e.g., FAM or FITC) on the 5′ end and a quencher on the 3′ end. The quencher (14) and fluorophore (16) typically are about 20-30 bases apart or less for effective quenching via fluorescence resonance energy transfer (FRET). Reporter moieties (12) are described in greater detail below. As more activated ribonucleoprotein complexes (6) are activated (6→8), more trans-cleavage activity of the nucleic acid-guided nuclease in the ribonucleoprotein complex is activated and more reporter moieties are activated (where here, “activated” means unquenched); thus, the binding of the target nucleic acid of interest (4).

As noted above, the downside to the prior art, currently available state-of-the-art nucleic acid-guided nuclease detection assays is that these detection assays rely on DNA amplification, which, in addition to issues with multiplexing, significantly hinders the ability to perform rapid point-of-care testing. The lack of rapidity is due to cis-cleavage of a target nucleic acid of interest being a single turnover event in which the number of activated enzyme complexes is, at most, equal to the number of copies of the target nucleic acids of interest in the sample. Once the ribonucleoprotein complex is activated after completion of cis-cleavage, trans-cleavage activity of the reporter moieties that are initially quenched is generated. However, the K_(cat) of, e.g., activated Cas12a complex is 17/sec and 3/sec for dsDNA and ssDNA targets, respectively. Therefore, for less than 10,000 target copies, the number of reporters cleaved is not sufficient to generate a signal in less than 30-60 minutes.

FIG. 1B provides a simplified diagram demonstrating a method (100) of a nucleic acid-guided nuclease cascade assay. The cascade assay is initiated when the target nucleic acid of interest (104) binds to and activates a first pre-assembled ribonucleoprotein complex (RNP1) (102). A ribonucleoprotein complex comprises a guide RNA (gRNA) and a nucleic acid-guided nuclease, where the gRNA is integrated with the nucleic acid-guided nuclease. The gRNA, which includes a sequence complementary to the target nucleic acid of interest, guides an RNP complex to the target nucleic acid of interest and hybridizes to it. Typically, preassembled RNP complexes are employed in the reaction mix—as opposed to separate nucleic acid-guided nucleases and gRNAs—to facilitate rapid detection of the target nucleic acid(s) of interest.

“Activation” of RNP1 refers to activating trans-cleavage activity of the nucleic acid-guided nuclease in RNP1 (106) by first initiating cis-cleavage where the target nucleic acid of interest is cut by the nucleic acid-guided nuclease. The cis-cleavage activity initiates trans-cleavage activity (i.e., multi-turnover activity) of the nucleic acid-guided nuclease, where trans-cleavage is indiscriminate, non-sequence-specific cutting of nucleic acid molecules by the nucleic acid-guided nuclease of RNP1 (102). This trans-cleavage activity triggers activation of blocked ribonucleoprotein complexes (RNP2s) (108) in various ways, which are described in detail below. Each newly activated RNP2 (110) activates more RNP2 (108→110), which in turn cleave reporter moieties (112). The reporter moieties (112) may be a synthetic molecule linked or conjugated to a quencher (114) and a fluorophore (116) such as, for example, a probe with a dye label (e.g., FAM or FITC) on the 5′ end and a quencher on the 3′ end. The quencher (114) and fluorophore (116) can be about 20-30 bases apart or less for effective quenching via fluorescence resonance energy transfer (FRET). Reporter moieties also are described in greater detail below. As more RNP2s are activated (108→110), more trans-cleavage activity is activated and more reporter moieties are activated (where here, “activated” means unquenched); thus, the binding of the target nucleic acid of interest (104) to RNP1 (102) initiates what becomes a cascade of signal production (120), which increases exponentially. The cascade assay thus comprises a single turnover event that triggers a multi-turnover event that then triggers another multi-turnover event. As described below in relation to FIG. 4 , the reporter moieties (112) may be provided as molecules that are separate from the other components of the nucleic acid-guided nuclease cascade assay, or the reporter moieties may be covalently or non-covalently linked to the blocked nucleic acid molecules or synthesized activating molecules (i.e., the target molecules for the RNP2). The various components common to the embodiments of the cascade assay and methods described herein are described below.

Target Nucleic Acids of Interest

The target nucleic acid of interest may be a DNA, RNA, or cDNA molecule. Target nucleic acids of interest may be isolated from a sample or organism by standard laboratory techniques or may be synthesized by standard laboratory techniques (e.g., RT-PCR). In some embodiments, the target nucleic acids of interest are identified in a sample, such as a biological sample from a subject or an environmental sample (e.g., water or soil). Non-limiting examples of biological samples include blood, serum, plasma, saliva, mucus, a nasal swab, a buccal swab, a cell, a cell culture, and tissue. The source of the sample could be any mammal, such as, but not limited to, a human, primate, monkey, cat, dog, mouse, pig, cow, horse, sheep, and bat. Samples may also be obtained from any other source, such as air, water, soil, surfaces, food, beverages, nutraceuticals, clinical sites or products, industrial sites and products, cosmetics, personal care products, pharmaceuticals, medical devices, agricultural equipment and sites, and commercial samples.

In some embodiments, the target nucleic acid of interest is from an infectious agent (e.g., a bacteria, protozoan, insect, worm, virus, or fungus). As a non-limiting example, the target nucleic acid of interest could be one or more nucleic acid molecules from bacteria, such as Bordetella parapertussis, Bordetella pertussis, Chlamydia pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae, Acinetobacter calcoaceticus-baumannii complex, Bacteroides fragilis, Enterobacter cloacae complex, Escherichia coli, Klebsiella aerogenes, Klebsiella oxytoca, Klebsiella pneumoniae group, Moraxella catarrhalis, Proteus spp., Salmonella enterica, Serratia marcescens, Haemophilus influenzae, Neisseria meningitidis, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Enterococcus faecalis, Enterococcus faecium, Listeria monocytogenes, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lugdunensis, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Chlamydia tracomatis, Neisseria gonorrhoeae, Syphilis (Treponema pallidum), Ureaplasma urealyticum, Mycoplasma genitalium, and/or Gardnerella vaginalis. As a non-limiting example, the target nucleic acid of interest could be one or more nucleic acid molecules from a virus, such as adenovirus, coronavirus HKU1, coronavirus NL63, coronavirus 229E, coronavirus OC43, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human metapneumovirus, human rhinovirus, enterovirus, influenza A, influenza A/H1, influenza A/H3, influenza A/H1-2009, influenza B, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4, respiratory syncytial virus, herpes simplex virus 1, herpes simplex virus 2, human immunodeficiency virus (HIV), human papillomavirus, hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), and/or human parvovirus B19 (B19V). Also, as a non-limiting example, the target nucleic acid of interest could be one or more nucleic acid molecules from a fungus, such as Candida albicans, Candida auris, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis, Cryptococcus neoformans, and/or Cryptococcus gattii. As another non-limiting example, the target nucleic acid of interest could be one or more nucleic acid molecules from a protozoan, such as Trichomonas vaginalis. In some embodiments, other target nucleic acids of interest may be for non-infectious conditions, e.g., to be used for genotyping. Other target nucleic acids of interest and samples are described herein.

The cascade assays described herein are particularly well-suited for syndromic testing. Syndromic testing allows simultaneous testing for multiple causative agents that cause similar symptoms. Syndromic testing allows rapid triage of patients, such as those needing emergency care, those amenable to treatment with pharmaceutical agents, those needing to be quarantined, etc. In syndrome testing, multiple target nucleic acids of interest are pooled into a single reaction, and this process may be repeated in multiple, separate reactions. A positive result in one of the reactions indicates that one of the target nucleic acids of interest in that pool is present. Pools of two to 10,000 target nucleic acids of interest may be employed, e.g., 2-1000, 2-100, 2-50, or 2-10. Further testing may be used to identify the specific member of the pool, if warranted. Syndromic testing allows the rapid triage of patients with the ability to focus further care rapidly.

While the methods described herein do not require the target nucleic acid of interest to be DNA (and in fact it is specifically contemplated that the target nucleic acid of interest may be RNA), it is understood by those in the field that a reverse transcription step to convert target RNA to cDNA may be performed prior to or while contacting the biological sample with the composition.

Nucleic Acid-Guided Nucleases

The cascade assays comprise nucleic acid-guided nucleases in the reaction mix, either provided as a protein, a coding sequence for the protein, or in a ribonucleoprotein (RNP) complex. In some embodiments, the one or more nucleic acid-guided nucleases in the reaction mix may be, for example, a Cas endonuclease. Any nucleic acid-guided nuclease having both cis- and trans-endonuclease activity may be employed, and the same nucleic acid-guided nuclease may be used for both RNPs or different nucleic acid-guided nucleases may be used in RNP1 and RNP2. Note that trans-cleavage activity is not triggered unless and until cis-cleavage activity (i.e., sequence specific activity) is initiated. Nucleic acid-guided nucleases include Type V and Type VI nucleic acid-guided nucleases, as well as nucleic acid-guided nucleases that comprise a RuvC nuclease domain or a RuvC-like nuclease domain but lack an HNH nuclease domain. Nucleic acid-guided nucleases with these properties are reviewed in Makarova and Koonin, Methods Mol. Biol., 1311:47-75 (2015) and Koonin, et al., Current Opinion in Microbiology, 37:67-78 (2020) and updated databases of nucleic acid-guided nucleases and nuclease systems that include newly-discovered systems include BioGRID ORCS (orcs:thebiogrid.org); GenomeCRISPR (genomecrispr.org); Plant Genome Editing Database (plantcrispr.org) and CRISPRCasFinder (crispercas.i2bc.paris-saclay.fr).

The type of nucleic acid-guided nuclease utilized in the method of detection depends on the type of target nucleic acid of interest to be detected. For example, a DNA nucleic acid-guided nuclease (e.g., a Cas12a, Cas14a, or Cas3) should be utilized if the target nucleic acid of interest is a DNA molecule, and an RNA nucleic acid-guided nuclease (e.g., Cas13a or Cas12g) should be utilized if the target nucleic acid of interest is an RNA molecule. Exemplary nucleic acid-guided nucleases include, but are not limited to, Cas RNA-guided DNA endonucleases, such as Cas3, Cas12a (e.g., AsCas12a, LbCas12a), Cas12b, Cas12c, Cas12d, Cas12e, Cas14, Cas12h, Cas12i, and Cas12j; Cas RNA-guided RNA endonucleases, such as Cas13a (LbaCas13, LbuCas13, LwaCas13), Cas13b (e.g., CccaCas13b, PsmCas13b), and Cas12g; and any other nucleic acid (DNA, RNA, or cDNA) targeting nucleic acid-guided nuclease with cis-cleavage activity and collateral trans-cleavage activity. In some embodiments, the nucleic acid-guided nuclease is a Type V CRISPR-Cas nuclease, such as a Cas12a, Cas13a, or Cas14a. In some embodiments, the nucleic acid-guided nuclease is a Type I CRISPR-Cas nuclease, such as Cas3. Type II and Type VI nucleic acid-guided nucleases may also be employed.

Guide RNA (gRNA)

The present disclosure detects a target nucleic acid of interest via a reaction mixture containing at least two gRNAs. Suitable guide RNAs include at least one crRNA region to enable specificity in every reaction. The gRNA of RNP1 is specific to a target nucleic acid of interest, and the gRNA of RNP2 is specific to an unblocked nucleic acid or a synthesized activating molecule (both described in detail herein). As will be clear given the description below, an advantageous feature of the cascade assay is that, with the exception of the gRNA in the RNP1 (i.e., the gRNA specific to the target nucleic acid of interest), the cascade assay components can stay the same no matter what target nucleic acid(s) of interest are being detected. In this sense, the cascade assay is modular.

Like the nucleic acid-guided nuclease, the gRNA may be provided in the cascade assay reaction mix in a preassembled RNP, as an RNA molecule, or may also be provided as a DNA sequence to be transcribed, in, e.g., a vector backbone. If provided as a gRNA molecule, the gRNA sequence may include multiple endoribonuclease recognition sites (e.g., Csy4) for multiplex processing. Alternatively, if provided as a DNA sequence to be transcribed, an endoribonuclease recognition site is encoded between neighboring gRNA sequences and more than one gRNA can be transcribed in a single expression cassette. Direct repeats can also serve as endoribonuclease recognition sites for multiplex processing. Guide RNAs are generally about 20 nucleotides to about 300 nucleotides in length and may contain a spacer sequence containing a plurality of bases and complementary to a protospacer sequence in the target sequence. The gRNA spacer sequence may be 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or more complementary to its intended target nucleic acid of interest.

The gRNA of RNP1 is capable of complexing with the nucleic acid-guided nuclease to perform cis-cleavage of a target nucleic acid of interest (e.g., a DNA or RNA), which triggers non-sequence specific trans-cleavage of other molecules in the reaction mix. Guide RNAs include any polynucleotide sequence having sufficient complementarity with a target nucleic acid of interest (or target sequences generated by unblocking blocked nucleic acid molecules or target sequences generated by synthesizing activating molecules as described below). Target sequences may include a protospacer-adjacent motif (PAM), and, following gRNA binding, the nucleic acid-guided nuclease induces a double-stranded break either inside or outside the protospacer region of the target sequence.

In some embodiments, the gRNA (e.g., of RNP1) is an exo-resistant circular molecule that can include several DNA bases between the 5′ end and the 3′ end of a natural guide RNA and is capable of binding a target sequence. The length of the circularized guide for RNP1 can be such that the circular form of guide can be complexed with a nucleic acid-guided nuclease to form a modified RNP1 which can still retain its cis-cleavage (specific) and trans-cleavage (non-specific) nuclease activity.

In any of the foregoing embodiments, the gRNA may be a modified or non-naturally occurring nucleic acid molecule. In some embodiments, the gRNAs of the disclosure may further contain a locked nucleic acid (LNA), a bridged nucleic acid (BNA), and/or a peptide nucleic acid (PNA). By way of further example, a modified nucleic acid molecule may contain a modified or non-naturally occurring nucleoside, nucleotide, and/or internucleoside linkage, such as a 2′-O-methyl (2′-O-Me) modified nucleoside, a 2′-fluoro (2′-F) modified nucleoside, and a phosphorothioate (PS) bond, or any other nucleic acid molecule modifications described herein.

Ribonucleoprotein (RNP) Complex

As described above, although the assay “reaction mix” may comprise separate nucleic acid-guided nucleases and gRNAs (or coding sequences therefor), the cascade assays preferably comprise preassembled ribonucleoprotein complexes (RNPs) in the reaction mix, allowing for faster detection kinetics. The present cascade assay employs at least two types of RNP complexes, RNP1 and RNP2, each type containing a nucleic acid-guided nuclease and a gRNA. RNP1 and RNP2 may comprise the same nucleic acid-guided nuclease or may comprise different nucleic acid-guided nucleases; however, the gRNAs in RNP1 and RNP2 are different and are configured to detect different nucleic acids. In some embodiments, the reaction mixture contains about 1 fM to about 10 μM of a given RNP1, or about 1 pM to about 1 μM of a given RNP1, or about 10 pM to about 500 pM of a given RNP1. In some embodiments the reaction mixture contains about 6×10⁴ to about 6×10¹² complexes per microliter (μl) of a given RNP1, or about 6×10⁶ to about 6×10¹⁰ complexes per microliter (μl) of a given RNP1. In some embodiments, the reaction mixture contains about 1 fM to about 1 mM of a given RNP2, or about 1 pM to about 500 μM of a given RNP2, or about 10 pM to about 100 μM of a given RNP2. In some embodiments the reaction mixture contains about 6×10⁴ to about 6×10¹⁴ complexes per microliter (μl) of a given RNP2 or about 6×10⁶ to about 6×10¹² complexes per microliter (μl) of a given RNP2. (See Example II below describing preassembling RNPs and Examples V-IX below describing various cascade assay conditions, including performing the cascade assay at room temperature.)

In any of the embodiments of the disclosure, the reaction mixture includes 1 to about 1,000 different RNP1s (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 27, 28, 19, 20, 21, 22, 23, 24, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1,0000 RNP1s), where different RNP1s comprise a different gRNA (or crRNA thereof) polynucleotide sequence. For example, a reaction mixture designed for syndromic testing by definition comprises more than one unique RNP1-gRNA (or RNP1-crRNA) ribonucleoprotein complex for the purpose of detecting more than one target nucleic acid of interest. More than one RNP1 may also be present for the purpose of targeting more than one target nucleic acid of interest from a single organism or condition.

In any of the foregoing embodiments, the gRNA of RNP1 may be homologous or heterologous, relative to the gRNA of other RNP1 present in the reaction mixture. A homologous mixture of RNP1 gRNAs has a number of gRNAs with the same nucleotide sequence, whereas a heterologous mixture of RNP1 gRNAs has multiple gRNAs with different nucleotide sequences (e.g., gRNAs targeting different loci, genes, variants, and/or microbial species). Therefore, the disclosed methods of identifying one or more target nucleic acids of interest may include a reaction mixture containing more than two heterologous gRNAs, more than three heterologous gRNAs, more than four heterologous gRNAs, more than five heterologous gRNAs, more than six heterologous gRNAs, more than seven heterologous gRNAs, more than eight heterologous gRNAs, more than nine heterologous gRNAs, more than ten heterologous gRNAs, more than eleven heterologous gRNAs, more than twelve heterologous gRNAs, more than thirteen heterologous gRNAs, more than fourteen heterologous gRNAs, more than fifteen heterologous gRNAs, more than sixteen heterologous gRNAs, more than seventeen heterologous gRNAs, more than eighteen heterologous gRNAs, more than nineteen heterologous gRNAs, more than twenty heterologous gRNAs, more than twenty-one heterologous gRNAs, more than twenty-three heterologous gRNAs, more than twenty-four heterologous gRNAs, or more than twenty-five heterologous gRNAs. Such a heterologous mixture of RNP1 gRNAs in a single reaction enables the capability of syndromic testing.

As a first non-limiting example of a heterologous mixture of RNP1 gRNAs, the reaction mixture may contain: a number of RNP1s having a gRNA targeting parainfluenza virus 1; a number of RNP1s having a gRNA targeting human metapneumovirus; a number of RNP1s having a gRNA targeting human rhinovirus; a number of RNP1s having a gRNA targeting human enterovirus; and a number of RNP1s having a gRNA targeting coronavirus HKU1. As a second non-limiting example of a heterologous mixture of RNP1 gRNAs, the reaction mixture may contain: a number of RNP1s containing a gRNA targeting two or more SARS-Co-V-2 variants, e.g., B.1.1.7, B.1.351, P.1, B.1.617.2, BA.1, BA.2, BA.2.12.1, BA.4, and BA.5 and subvariants thereof.

Reporter Moieties

The cascade assay detects a target nucleic acid of interest via detection of a signal generated in the reaction mix by a reporter moiety. In some embodiments the detection of the target nucleic acid of interest occurs in about 10 minutes or less (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 minute or less; e.g., FIGS. 6-9 , and in some embodiments the detection of the target nucleic acid molecule is in about 5 minutes or less (e.g., 5, 4, 3, 2, or 1 minute or less; e.g., FIGS. 10-14 ). In some embodiments, the detection of the target nucleic acid molecule is in about 1 minute (e.g., FIGS. 10-13 ).

Depending on the type of reporter moiety used, trans- and/or cis-cleavage by the nucleic acid-guided nuclease in RNP2 releases a signal. In some embodiments, trans-cleavage of stand-alone (e.g., not bound to any blocked nucleic acid molecules) reporter moieties may generate signal changes at rates that are proportional to the cleavage rate, as new RNP2s are activated over time (shown in FIG. 1B and at top of FIG. 4 ). Trans-cleavage by either an activated RNP1 or an activated RNP2 may release a signal. In alternative embodiments, the reporter moiety may be bound to the blocked nucleic acid molecule, where trans-cleavage of the blocked nucleic acid molecule and conversion to an unblocked nucleic acid molecule may generate signal changes at rates that are proportional to the cleavage rate, as new RNP2s are activated over time, thus allowing for real time reporting of results (shown at FIG. 4 , center). In yet another embodiment, the reporter moiety may be bound to a blocked nucleic acid molecule such that cis-cleavage following the binding of the RNP2 to an unblocked nucleic acid molecule releases a PAM distal sequence, which in turn generates a signal at rates that are proportional to the cleavage rate (shown at FIG. 4 , bottom). In this case, activation of RNP2 by cis-(target specific) cleavage of the unblocked nucleic acid molecule directly produces a signal, rather than producing a signal via indiscriminate trans-cleavage activity. Alternatively. or in addition, the reporter moiety may be bound to the gRNA.

The reporter moiety may be a synthetic molecule linked or conjugated to a reporter and quencher such as, for example, a TaqMan probe with a dye label (e.g., FAM or FITC) on the 5′ end and a quencher on the 3′ end. The reporter and quencher may be about 20-30 bases apart or less for effective quenching via fluorescence resonance energy transfer (FRET). Alternatively, signal generation may occur through different mechanisms. Other detectable moieties, labels, or reporters can also be used to detect a target nucleic acid of interest as described herein. Reporter moieties can be labeled in a variety of ways, including direct or indirect attachment of a detectable moiety such as a fluorescent moiety, hapten, or colorimetric moiety. Examples of detectable moieties include various radioactive moieties, enzymes, prosthetic groups, fluorescent markers, luminescent markers, bioluminescent markers, metal particles, and protein-protein binding pairs, e.g., protein-antibody binding pairs. Examples of fluorescent moieties include, but are not limited to, yellow fluorescent protein (YFP), green fluorescence protein (GFP), cyan fluorescence protein (CFP), umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, cyanines, dansyl chloride, phycocyanin, and phycoerythrin. Examples of bioluminescent markers include, but are not limited to, luciferase (e.g., bacterial, firefly, click beetle and the like), luciferin, and aequorin. Examples of enzyme systems having visually detectable signals include, but are not limited to, galactosidases, glucuronidases, phosphatases, peroxidases, and cholinesterases. Identifiable markers also include radioactive elements such as ¹²⁵1, ³⁵S, ¹⁴C, or ³H.

The methods used to detect the generated signal will depend on the reporter moiety or moieties used. For example, a radioactive label can be detected using a scintillation counter, photographic film as in autoradiography, or storage phosphor imaging. Fluorescent labels can be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence can be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like. Enzymatic labels can be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product. Simple colorimetric labels can be detected by observing the color associated with the label. When pairs of fluorophores are used in an assay, fluorophores are chosen that have distinct emission patterns (wavelengths) so that they can be easily distinguished. In some embodiments, the signal can be detected by lateral flow assays (LFAs). Lateral flow tests are simple devices intended to detect the presence or absence of a target nucleic acid of interest in a sample. LFAs can use nucleic acid molecules conjugated nanoparticles (often gold, e.g., RNA-AuNPs or DNA-AuNPs) as a detection probe, which hybridizes to a complementary target sequence. (See FIGS. 5A and 5B and the description thereof below.) The classic example of an LFA is the home pregnancy test.

Single-stranded nucleic acid reporter moieties such as ssDNA reporter moieties or RNA molecules can be introduced to show a signal change proportional to the cleavage rate, which increases with every new activated RNP2 complex over time. In some embodiments and as described in detail below, single-stranded nucleic acid reporter moieties can also be embedded into the blocked nucleic acid molecules for real time reporting of results.

For example, the method of detecting a target nucleic acid molecule in a sample using a cascade assay as described herein can involve contacting the reaction mix with a labeled detection ssDNA containing a fluorescent resonance energy transfer (FRET) pair, a quencher/phosphor pair, or both. A FRET pair consists of a donor chromophore and an acceptor chromophore, where the acceptor chromophore may be a quencher molecule. FRET pairs (donor/acceptor) suitable for use include, but are not limited to, EDANS/fluorescein, IAEDANS/fluorescein, fluorescein/tetramethylrhodamine, fluorescein/Cy 5, IEDANS/DABCYL, fluorescein/QSY-7, fluorescein/LC Red 640, fluorescein/Cy 5.5, Texas Red/DABCYL, BODIPY/DABCYL, Lucifer yellow/DABCYL, coumarin/DABCYL, and fluorescein/LC Red 705. In addition, a fluorophore/quantum dot donor/acceptor pair can be used. EDANS is (5-((2-Aminoethyl)amino)naphthalene-1-sulfonic acid); IAEDANS is 5-({2-[(iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid); DABCYL is 4-(4-dimethylaminophenyl)diazenylbenzoic acid. Useful quenchers include, but are not limited to, DABCYL, QSY 7 and QSY 33.

In any of the foregoing embodiments, the reporter moiety may comprise one or more modified nucleic acid molecules, containing a modified nucleoside or nucleotide. In some embodiments the modified nucleoside or nucleotide is chosen from 2′-O-methyl (2′-O-Me) modified nucleoside, a 2′-fluoro (2′-F) modified nucleoside, and a phosphorothioate (PS) bond, or any other nucleic acid molecule modifications described below.

Nucleic Acid Modifications

For any of the nucleic acid molecules described herein (e.g., blocked nucleic acid molecules, blocked primer molecules, gRNAs, template molecules, synthesized activating molecules, and reporter moieties), the nucleic acid molecules may be used in a wholly or partially modified form. Typically, modifications to the blocked nucleic acids, gRNAs, template molecules, reporter moieties, and blocked primer molecules described herein are introduced to optimize the molecule's biophysical properties (e.g., increasing endonuclease resistance and/or increasing thermal stability). Modifications typically are achieved by the incorporation of, for example, one or more alternative nucleosides, alternative sugar moieties, and/or alternative internucleoside linkages.

For example, one or more of the cascade assay components may include one or more of the following nucleoside modifications: 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C═C—CH₃) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and/or 3-deazaguanine and 3-deazaadenine. The nucleic acid molecules described herein (e.g., blocked nucleic acid molecules, blocked primer molecules, gRNAs, reporter molecules, synthesized activating molecules, and template molecules) may also include nucleobases in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and/or 2-pyridone. Further modification of the nucleic acid molecules described herein may include nucleobases disclosed in U.S. Pat. No. 3,687,808; Kroschwitz, ed. The Concise Encyclopedia of Polymer Science and Engineering, New York, John Wiley & Sons, 1990, pp. 858-859; Englisch, et al., Angewandte Chemie, 30:613 (1991); and Sanghvi, Chapter 16, Antisense Research and Applications, CRC Press, Gait, ed., 1993, pp. 289-302.

In addition to or as an alternative to nucleoside modifications, the cascade assay components may comprise 2′ sugar modifications, including 2′-O-methyl (2′-O-Me), 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE), 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, and/or 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylamino-ethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O—CH₂OCH₂N(CH₃)₂. Other possible 2′-modifications that can modify the nucleic acid molecules described herein (i.e., blocked nucleic acids, gRNAs, synthesized activating molecules, reporter molecules, and blocked primer molecules) may include all possible orientations of OH; F; O-, S-, or N-alkyl (mono- or di-); O-, S-, or N-alkenyl (mono- or di-); O-, S- or N-alkynyl (mono- or di-); or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Other potential sugar substituent groups include, e.g., aminopropoxy (—OCH₂CH₂CH₂NH₂), allyl (—CH₂—CH═CH₂), —O-allyl (—O—CH₂—CH═CH₂) and fluoro (F). 2′-sugar substituent groups may be in the arabino (up) position or ribo (down) position. In some embodiments, the 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the interfering RNA molecule, particularly the 3′ position of the sugar on the 3′ terminal nucleoside or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

Finally, modifications to the cascade assay components may comprise internucleoside modifications such as phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.

The Cascade Assay Employing Blocked Nucleic Acids

FIG. 1B depicts the cascade assay generally. A specific embodiment of the cascade assay utilizing blocked nucleic acids is depicted in FIG. 2A. In this embodiment, a blocked nucleic acid is used to prevent the activation of RNP2 in the absence of a target nucleic acid of interest. The method in FIG. 2A begins with providing the cascade assay components RNP1 (201), RNP2 (202) and blocked nucleic acid molecules (203). RNP1 (201) comprises a gRNA specific for a target nucleic acid of interest and a nucleic acid-guided nuclease (e.g., Cas 12a or Cas 14 for a DNA target nucleic acid of interest or a Cas 13a for an RNA target nucleic acid of interest) and RNP2 (202) comprises a gRNA specific for an unblocked nucleic acid molecule and a nucleic acid-guided nuclease (again, Cas 12a or Cas 14 for a DNA unblocked nucleic acid molecule or a Cas 13a for an RNA unblocked nucleic acid molecule). As described above, the nucleic acid-guided nucleases in RNP1 (201) and RNP2 (202) can be the same or different depending on the type of target nucleic acid of interest and unblocked nucleic acid molecule. What is key, however, is that the nucleic acid-guided nucleases in RNP1 and RNP2 may be activated to have trans-cleavage activity following initiation of cis-cleavage activity.

In a first step, a sample comprising a target nucleic acid of interest (204) is added to the cascade assay reaction mix. The target nucleic acid of interest (204) combines with and activates RNP1 (205) but does not interact with or activate RNP2 (202). Once activated, RNP1 cuts the target nucleic acid of interest (204) via sequence-specific cis-cleavage, which then activates non-specific trans-cleavage of other nucleic acids present in the reaction mix, including the blocked nucleic acid molecules (203). At least one of the blocked nucleic acid molecules (203) becomes an unblocked nucleic acid molecule (206) when the blocking moiety (207) is removed. As described below, “blocking moiety” may refer to nucleoside modifications, topographical configurations such as secondary structures, and/or structural modifications.

Once at least one of the blocked nucleic acid molecules (203) is unblocked, the unblocked nucleic acid molecule (206) can then interact with and activate an RNP2 (208) complex. Because the nucleic acid-guided nucleases in the RNP1x (205) and RNP2x (208) have both cis- and trans-cleavage activity, more blocked nucleic acid molecules (203) become unblocked nucleic acid molecules (206) triggering activation of more RNP2 (208) complexes and more trans-cleavage activity in a cascade. FIG. 2A at bottom depicts the concurrent activation of reporter moieties. Intact reporter moieties (209) comprise a quencher (210) and a fluorophore (211) linked by a nucleic acid sequence. As described above in relation to FIG. 1B, the reporter moieties are also subject to trans-cleavage by activated RNP1 (205) and RNP2 (208). The intact reporter moieties (209) become activated reporter moieties (212) when the quencher (210) is separated from the fluorophore (211), emitting a fluorescent signal (213). Signal strength increases rapidly as more blocked nucleic acid molecules (203) become unblocked nucleic acid molecules (206) triggering cis-cleavage activation of more RNP2s (208) and thus more trans-cleavage activity of the reporter moieties (209). Again, here the reporter moieties are shown as separate molecules from the blocked nucleic acid molecules, but other configurations may be employed and are discussed in relation to FIG. 4 . One particularly advantageous feature of the cascade assay is that, with the exception of the gRNA in the RNP1 (gRNA1), the cascade assay components are modular in the sense that the components stay the same no matter what target nucleic acid(s) of interest are being detected.

FIG. 2B is a diagram showing an exemplary blocked nucleic acid molecule (220) and an exemplary technique for unblocking the blocked nucleic acid molecules described herein. A blocked single-stranded or double-stranded, circular or linear, DNA or RNA molecule (220) comprising a target strand (222) may contain a partial hybridization with a complementary non-target strand nucleic acid molecule (224) containing unhybridized and cleavable secondary loop structures (226) (e.g., hairpin loops, tetraloops, pseudoknots, junctions, kissing hairpins, internal loops, bulges, and multibranch loops). Trans-cleavage of the loops by, e.g., activated RNP1s or RNP2s, generates short strand nucleotide sequences (228) which, because of the short length and low melting temperature T_(m), can dehybridize at room temperature (e.g., 15°-25° C.), thereby unblocking the blocked nucleic acid molecule (220) to create an unblocked nucleic acid molecule (230), enabling the internalization of the unblocked nucleic acid molecule (230) (target strand) into an RNP2, leading to RNP2 activation.

A blocked nucleic acid molecule may be single-stranded or double-stranded, circular or linear, and may further contain a partially hybridized nucleic acid sequence containing cleavable secondary loop structures, as exemplified by “L” in FIGS. 2C-2E. Such blocked nucleic acids typically have a low binding affinity, or high dissociation constant (K_(d)) in relation to binding to RNP2 and may be referred to herein as a high K_(d) nucleic acid molecule. In the context of the present disclosure, the binding of blocked or unblocked nucleic acid molecules or blocked or unblocked primer molecules to RNP2, low K_(d) values range from about 100 fM to about 1 aM or lower (e.g., 100 zM) and high K_(d) values are in the range of 100 nM to about 100 μM (10 mM) and thus are about 10⁵-, 10⁶-, 10⁷-, 10⁸-, 10⁹- to 10¹⁰-fold or higher as compared to low K_(d) values.

The blocked nucleic acid molecules (high K_(d) molecules) described herein can be converted into unblocked nucleic acid molecules (low K_(d) molecules—also in relation to binding to RNP2) via cleavage of nuclease-cleavable regions (e.g., via active RNP1s and RNP2s). The unblocked nucleic acid molecule has a higher binding affinity for the gRNA in the RNP2 than does the blocked nucleic acid molecule, although there may be some “leakiness” where some blocked nucleic acid molecules are able to interact with the gRNA in the RNP2. However, an unblocked nucleic acid molecule has a substantially higher likelihood than a blocked nucleic acid molecule to hybridize with the gRNA of RNP2.

Once the unblocked nucleic acid molecule is bound to RNP2, the RNP2 activation triggers trans-cleavage activity, which in turn leads to more RNP2 activation by further cleaving blocked nucleic acid molecules, resulting in a positive feedback loop.

In embodiments where blocked nucleic acid molecules are linear and/or form a secondary structure, the blocked nucleic acid molecules may be single-stranded (ss) or double-stranded (ds) and contain a first nucleotide sequence and a second nucleotide sequence. The first nucleotide sequence has sufficient complementarity to hybridize to a gRNA of RNP2, and the second nucleotide sequence does not. The first and second nucleotide sequences of a blocked nucleic acid molecule may be on the same nucleic acid molecule (e.g., for single-strand embodiments) or on separate nucleic acid molecules (e.g., for double strand embodiments). Trans-cleavage (e.g., via RNP1 or RNP2) of the second nucleotide sequence converts the blocked nucleic acid molecule to a single-strand unblocked nucleic acid molecule. The unblocked nucleic acid molecule contains only the first nucleotide sequence, which has sufficient complementarity to hybridize to the gRNA of RNP2, thereby activating the trans-endonuclease activity of RNP2.

In some embodiments, the second nucleotide sequence at least partially hybridizes to the first nucleotide sequence, resulting in a secondary structure containing at least one loop (e.g., hairpin loops, tetraloops, pseudoknots, junctions, kissing hairpins, internal loops, bulges, and multibranch loops). Such loops block the nucleic acid molecule from binding or incorporating into an RNP complex in a manner to initiate trans cleavage (see, e.g., the exemplary structures in FIGS. 2C-2E).

In some embodiments, the blocked nucleic acid molecule may contain a protospacer adjacent motif (PAM) sequence, or partial PAM sequence, positioned between the first and second nucleotide sequences, where the first sequence is 5′ to the PAM sequence, or partial PAM sequence, (see FIG. 2G). Inclusion of a PAM sequence may increase the reaction kinetics internalizing the unblocked nucleic acid molecule into RNP2 and thus decrease the time to detection. In other embodiments, the blocked nucleic acid molecule does not contain a PAM sequence.

In some embodiments, the blocked nucleic acid molecules (i.e., high K_(d) nucleic acid molecules—in relation to binding to RNP2) of the disclosure may include a structure represented by Formula I (e.g., FIG. 2C), Formula II (e.g., FIG. 2D), Formula III (e.g., FIG. 2E), or Formula IV (e.g., FIG. 2F) wherein Formulas I-IV are in the 5′-to-3 direction:

A-(B-L)_(J)-C-M-T-D  (Formula I);

-   -   wherein A is 0-15 nucleotides in length;     -   B is 4-12 nucleotides in length;     -   L is 3-25 nucleotides in length;     -   J is an integer between 1 and 10;     -   C is 4-15 nucleotides in length;     -   M is 1-25 nucleotides in length or is absent, wherein if M is         absent then A-(B-L)_(J)-C and T-D are separate nucleic acid         strands;     -   T is 17-135 nucleotides in length (e.g., 17-100, 17-50, or         17-25) and comprises a sequence complementary to B and C; and     -   D is 0-10 nucleotides in length and comprises a sequence         complementary to A;

D-T-T′-C-(L-B)_(J)-A  (Formula II);

-   -   wherein D is 0-10 nucleotides in length;     -   T-T′ is 17-135 nucleotides in length (e.g., 17-100, 17-50, or         17-25);     -   T′ is 1-10 nucleotides in length and does not hybridize with T;     -   C is 4-15 nucleotides in length and comprises a sequence         complementary to T;     -   L is 3-25 nucleotides in length and does not hybridize with T;     -   B is 4-12 nucleotides in length and comprises a sequence         complementary to T;     -   J is an integer between 1 and 10;     -   A is 0-15 nucleotides in length and comprises a sequence         complementary to D;

T-D-M-A-(B-L)_(J)-C  (Formula III);

-   -   wherein T is 17-135 nucleotides in length (e.g., 17-100, 17-50,         or 17-25);     -   D is 0-10 nucleotides in length;     -   M is 1-25 nucleotides in length or is absent, wherein if M is         absent then T-D and A-(B-L)_(J)-C are separate nucleic acid         strands;     -   A is 0-15 nucleotides in length and comprises a sequence         complementary to D;     -   B is 4-12 nucleotides in length and comprises a sequence         complementary to T;     -   L is 3-25 nucleotides in length;     -   J is an integer between 1 and 10; and     -   C is 4-15 nucleotides in length;

T-D-M-A-L_(p)-C  (Formula IV);

-   -   wherein T is 17-31 nucleotides in length (e.g., 17-100, 17-50,         or 17-25);     -   D is 0-15 nucleotides in length;     -   M is 1-25 nucleotides in length;     -   A is 0-15 nucleotides in length and comprises a sequence         complementary to D; and     -   L is 3-25 nucleotides in length;     -   p is 0 or 1;     -   C is 4-15 nucleotides in length and comprises a sequence         complementary to T.         In alternative embodiments of any of these molecules, T (or         T-T′) can have a maximum length of 1000 nucleotides, e.g., at         most 200, at most 135, at most 75, at most 50, or at most 25.

Nucleotide mismatches can be introduced in any of the above structures containing double strand segments (for example, where M is absent in Formula I or Formula III) to reduce the melting temperature (Tm) of the segment such that once the loop (L) is cleaved, the double strand segment is unstable and dehybridizes rapidly. The percentage of nucleotide mismatches of a given segment may vary between 0% and 50%; however, the maximum number of nucleotide mismatches is limited to a number where the secondary loop structure still forms. “Segments” in the above statement refers to A, B, and C. In other words, the number of hybridized bases can be less than or equal to the length of each double strand segment and vary based on number of mismatches introduced.

In any blocked nucleic acid molecule having the structure of Formula I, III, or IV, T will have sequence complementarity to a nucleotide sequence (e.g., a spacer sequence) within a gRNA of RNP2. The nucleotide sequence of T is to be designed such that hybridization of T to the gRNA of RNP2 activates the trans-nuclease activity of RNP2. In any blocked nucleic acid molecule having structure of Formula II, T-T′ will have sequence complementarity to a sequence (e.g., a spacer sequence) within the gRNA of RNP2. The nucleotide sequence of T-T′ is to be designed such that hybridization of T-T′ to the gRNA of RNP2 activates the trans-nuclease activity of RNP2. For T or T-T′, full complementarity to the gRNA is not necessarily required, provided there is sufficient complementarity to cause hybridization and trans-cleavage activation of RNP2.

Exemplary nucleotide sequences of blocked nucleic acid molecules (e.g., SEQ ID NOs: 14-1421) include those in Table 1.

TABLE 1 Nucleotide sequences of blocked nucleic acid molecules. SEQ ID NO: Sequence SEQ ID NO: 14 GATACTTTTTATTTTTTATATAT ATATATATTTTTTATTTTTATA TATATATATATAGTATC SEQ ID NO: 15 GACACTTTTTATTTTTTATATAT ATATATATTTTTTATTTTTATA TATATATATATAGTGTC SEQ ID NO: 16 GATACTTTTTATTTTTGATATAT ATATATATTTTTTATTTTTATA TATATATATATCGTATC SEQ ID NO: 17 GGATCTTTTTATTTTTTATATAT ATATATATTTTTTATTTTTATA TATATATATATAGATCC SEQ ID NO: 18 GACACTTTTTATTTTTGATATAT ATATATATTTTTTATTTTTATA TATATATATATCGTGTC SEQ ID NO: 19 GGATCTTTTTATTTTTGATATAT ATATATATTTTTTATTTTTATA TATATATATATCGATCC SEQ ID NO: 20 GCGTCTTTTTATTTTTTATATAT ATATATATTTTTTATTTTTATA TATATATATATAGACGC SEQ ID NO: 21 GCGTCTTTTTATTTTTGATATAT ATATATATTTTTTATTTTTATA TATATATATATCGACGC SEQ ID NO: 22 GTATACTTTTTATTTTTTATATA TATATATATTTTTATTTTTTAT ATATATATATAGTATAC SEQ ID NO: 23 GTGATCTTTTTATTTTTTATATA TATATATATTTTTATTTTTTAT ATATATATATAGATCAC SEQ ID NO: 24 GTATACTTTTTATTTTTGATATA TATATATATTTTTATTTTTTAT ATATATATATCGTATAC SEQ ID NO: 25 GTATACTTTTTATTTTTGATATA TGTATATATTTTTATTTTTTAT ATACATATATCGTATAC SEQ ID NO: 26 GGATACTTTTTATTTTTTATATA TATATATATTTTTATTTTTTAT ATATATATATAGTATCC SEQ ID NO: 27 GTGATCTTTTTATTTTTGATATA TATATATATTTTTATTTTTTAT ATATATATATCGATCAC SEQ ID NO: 28 GTGATCTTTTTATTTTTGATATA TGTATATATTTTTATTTTTTAT ATACATATATCGATCAC SEQ ID NO: 29 GGATACTTTTTATTTTTGATATA TATATATATTTTTATTTTTTAT ATATATATATCGTATCC SEQ ID NO: 30 GGATACTTTTTATTTTTGATATA TGTATATATTTTTATTTTTTAT ATACATATATCGTATCC SEQ ID NO: 31 GCGATCTTTTTATTTTTTATATA TATATATATTTTTATTTTTTAT ATATATATATAGATCGC SEQ ID NO: 32 GCGATCTTTTTATTTTTGATATA TATATATATTTTTATTTTTTAT ATATATATATCGATCGC SEQ ID NO: 33 GCGATCTTTTTATTTTTGATATA TGTATATATTTTTATTTTTTAT ATACATATATCGATCGC SEQ ID NO: 34 GATATACTTTTTATTTTTTATAT ATATATATTTTTTATTTTTATA TATATATATAGTATATC SEQ ID NO: 35 GATATATTTTTTATTTTTGATAT ATATATATTTTTTATTTTTATA TATATATATCATATATC SEQ ID NO: 36 GATATATTTTTTATTTTTGATAT ATGTATATTTTTTATTTTTATA TACATATATCATATATC SEQ ID NO: 37 GTGATACTTTTTATTTTTTATAT ATATATATTTTTTATTTTTATA TATATATATAGTATCAC SEQ ID NO: 38 GATATACTTTTTATTTTTGATAT ATATATATTTTTTATTTTTATA TATATATATCGTATATC SEQ ID NO: 39 GATATACTTTTTATTTTTGATAT ATGTATATTTTTTATTTTTATA TACATATATCGTATATC SEQ ID NO: 40 GGTATACTTTTTATTTTTTATAT ATATATATTTTTTATTTTTATA TATATATATAGTATACC SEQ ID NO: 41 GTGATACTTTTTATTTTTGATAT ATATATATTTTTTATTTTTATA TATATATATCGTATCAC SEQ ID NO: 42 GTGATACTTTTTATTTTTGATAT ATGTATATTTTTTATTTTTATA TACATATATCGTATCAC SEQ ID NO: 43 GGTATACTTTTTATTTTTGATAT ATATATATTTTTTATTTTTATA TATATATATCGTATACC SEQ ID NO: 44 GGTATACTTTTTATTTTTGATAT ATGTATATTTTTTATTTTTATA TACATATATCGTATACC SEQ ID NO: 45 GGTGTACTTTTTATTTTTTATAT ATATATATTTTTTATTTTTATA TATATATATAGTACACC SEQ ID NO: 46 GGTGTACTTTTTATTTTTGATAT ATATATATTTTTTATTTTTATA TATATATATCGTACACC SEQ ID NO: 47 GGTGTACTTTTTATTTTTGATAT ATGTATATTTTTTATTTTTATA TACATATATCGTACACC SEQ ID NO: 48 GTATATACTTTTTATTTTTTATA TATATATATTTTTATTTTTTAT ATATATATAGTATATAC SEQ ID NO: 49 GTATATACTTTTTATTTTTGATA TATATATATTTTTATTTTTTAT ATATATATCGTATATAC SEQ ID NO: 50 GTATATACTTTTTATTTTTGATA TATGTATATTTTTATTTTTTAT ACATATATCGTATATAC SEQ ID NO: 51 GTATATACTTTTTATTTTTGATC ATGTATATTTTTTATTTTTATA TACATGATCGTATATAC SEQ ID NO: 52 GTATATACTTTTTATTTTTGATC ATATATGTTTTTTATTTTTACA TATATGATCGTATATAC SEQ ID NO: 53 GGATATACTTTTTATTTTTTATA TATATATATTTTTATTTTTTAT ATATATATAGTATATCC SEQ ID NO: 54 GGATATACTTTTTATTTTTGATA TATATATATTTTTATTTTTTAT ATATATATCGTATATCC SEQ ID NO: 55 GGATATACTTTTTATTTTTGATA TATGTATATTTTTATTTTTTAT ACATATATCGTATATCC SEQ ID NO: 56 GGATATACTTTTTATTTTTGATC ATGTATATTTTTTATTTTTATA TACATGATCGTATATCC SEQ ID NO: 57 GGATATACTTTTTATTTTTGATC ATATATGTTTTTTATTTTTACA TATATGATCGTATATCC SEQ ID NO: 58 GGTGATACTTTTTATTTTTTATA TATATATATTTTTATTTTTTAT ATATATATAGTATCACC SEQ ID NO: 59 GGTGATACTTTTTATTTTTGATA TATATATATTTTTATTTTTTAT ATATATATCGTATCACC SEQ ID NO: 60 GGTGATACTTTTTATTTTTGATA TATGTATATTTTTATTTTTTAT ACATATATCGTATCACC SEQ ID NO: 61 GGTGATACTTTTTATTTTTGATC ATGTATATTTTTTATTTTTATA TACATGATCGTATCACC SEQ ID NO: 62 GGTGATACTTTTTATTTTTGATC ATATATGTTTTTTATTTTTACA TATATGATCGTATCACC SEQ ID NO: 63 GGTGATCCTTTTTATTTTTTATA TATATATATTTTTATTTTTTAT ATATATATAGGATCACC SEQ ID NO: 64 GGTGATCCTTTTTATTTTTGATA TATATATATTTTTATTTTTTAT ATATATATCGGATCACC SEQ ID NO: 65 GGTGATCCTTTTTATTTTTGATA TATGTATATTTTTATTTTTTAT ACATATATCGGATCACC SEQ ID NO: 66 GGTGATCCTTTTTATTTTTGATC ATGTATATTTTTTATTTTTATA TACATGATCGGATCACC SEQ ID NO: 67 GGTGATCCTTTTTATTTTTGATC ATATATGTTTTTTATTTTTACA TATATGATCGGATCACC SEQ ID NO: 68 GATATATCACTTTTTATTTTTTA TATATATATTTTTATTTTTTAT ATATATAGTGATATATC SEQ ID NO: 69 GTATATACATTTTTTATTTTTGA TATATATATTTTTATTTTTTAT ATATATCATGTATATAC SEQ ID NO: 70 GTATATACATTTTTTATTTTTGA TATATGTATTTTTATTTTTTAC ATATATCATGTATATAC SEQ ID NO: 71 GTATATACATTTTTTATTTTTGA TCATGTATTTTTTATTTTTATA CATGATCATGTATATAC SEQ ID NO: 72 GTATATACATTTTTTATTTTTGA TCATATATTTTTTATTTTTATA TATGATCATGTATATAC SEQ ID NO: 73 GGATATACACTTTTTATTTTTTA TATATATATTTTTATTTTTTAT ATATATAGTGTATATCC SEQ ID NO: 74 GGATATACATTTTTTATTTTTGA TATATATATTTTTATTTTTTAT ATATATCATGTATATCC SEQ ID NO: 75 GGATATACATTTTTTATTTTTGA TATATGTATTTTTATTTTTTAC ATATATCATGTATATCC SEQ ID NO: 76 GGATATACATTTTTTATTTTTGA TCATGTATTTTTTATTTTTATA CATGATCATGTATATCC SEQ ID NO: 77 GGATATACATTTTTTATTTTTGA TCATATATTTTTTATTTTTATA TATGATCATGTATATCC SEQ ID NO: 78 GGGTATATACTTTTTATTTTTTA TATATATATTTTTATTTTTTAT ATATATAGTATATACCC SEQ ID NO: 79 GGATATACACTTTTTATTTTTGA TATATATATTTTTATTTTTTAT ATATATCGTGTATATCC SEQ ID NO: 80 GGATATACACTTTTTATTTTTGA TATATGTATTTTTATTTTTTAC ATATATCGTGTATATCC SEQ ID NO: 81 GGATATACACTTTTTATTTTTGA TCATGTATTTTTTATTTTTATA CATGATCGTGTATATCC SEQ ID NO: 82 GGATATACACTTTTTATTTTTGA TCATATATTTTTTATTTTTATA TATGATCGTGTATATCC SEQ ID NO: 83 GGGTATATACTTTTTATTTTTGA TATATATATTTTTATTTTTTAT ATATATCGTATATACCC SEQ ID NO: 84 GGGTATATACTTTTTATTTTTGA TATATGTATTTTTATTTTTTAC ATATATCGTATATACCC SEQ ID NO: 85 GGGTATATACTTTTTATTTTTGA TCATGTATTTTTTATTTTTATA CATGATCGTATATACCC SEQ ID NO: 86 GGGTATATACTTTTTATTTTTGA TCATATATTTTTTATTTTTATA TATGATCGTATATACCC SEQ ID NO: 87 GGATGTACACTTTTTATTTTTTA TATATATATTTTTATTTTTTAT ATATATAGTGTACATCC SEQ ID NO: 88 GGATGTACACTTTTTATTTTTGA TATATATATTTTTATTTTTTAT ATATATCGTGTACATCC SEQ ID NO: 89 GGATGTACACTTTTTATTTTTGA TATATGTATTTTTATTTTTTAC ATATATCGTGTACATCC SEQ ID NO: 90 GGATGTACACTTTTTATTTTTGA TCATGTATTTTTTATTTTTATA CATGATCGTGTACATCC SEQ ID NO: 91 GGATGTACACTTTTTATTTTTGA TCATATATTTTTTATTTTTATA TATGATCGTGTACATCC SEQ ID NO: 92 GTATATACTTTTTATTTTTTATA TATATATATATTTTTTATTTTT ATATATATATATATAGTATATAC SEQ ID NO: 93 GTATATACTTTTTATTTTTGATA TATATATATATTTTTTATTTTT ATATATATATATATCGTATATAC SEQ ID NO: 94 GGATATACTTTTTATTTTTTATA TATATATATATTTTTTATTTTT ATATATATATATATAGTATATCC SEQ ID NO: 95 GGATATACTTTTTATTTTTGATA TATATATATATTTTTTATTTTT ATATATATATATATCGTATATCC SEQ ID NO: 96 GGTGATACTTTTTATTTTTTATA TATATATATATTTTTTATTTTT ATATATATATATATAGTATCACC SEQ ID NO: 97 GGTGATACTTTTTATTTTTGATA TATATATATATTTTTTATTTTT ATATATATATATATCGTATCACC SEQ ID NO: 98 GGTGATCCTTTTTATTTTTTATA TATATATATATTTTTTATTTTT ATATATATATATATAGGATCACC SEQ ID NO: 99 GGTGATCCTTTTTATTTTTGATA TATATATATATTTTTTATTTTT ATATATATATATATCGGATCACC SEQ ID NO: 100 GATATATCACTTTTTATTTTTTA TATATATATATATTTTTTATTT TTATATATATATATATAGTGATA TATC SEQ ID NO: 101 GTATATACATTTTTTATTTTTGA TATATATATATATTTTTTATTT TTATATATATATATATCATGTAT ATAC SEQ ID NO: 102 GGATATACACTTTTTATTTTTTA TATATATATATATTTTTTATTT TTATATATATATATATAGTGTAT ATCC SEQ ID NO: 103 GGATATACATTTTTTATTTTTGA TATATATATATATTTTTTATTT TTATATATATATATATCATGTAT ATCC SEQ ID NO: 104 GGGTATATACTTTTTATTTTTTA TATATATATATATTTTTTATTT TTATATATATATATATAGTATAT ACCC SEQ ID NO: 105 GGATATACACTTTTTATTTTTGA TATATATATATATTTTTTATTT TTATATATATATATATCGTGTAT ATCC SEQ ID NO: 106 GGGTATATACTTTTTATTTTTGA TATATATATATATTTTTTATTT TTATATATATATATATCGTATAT ACCC SEQ ID NO: 107 GTATATACTTTTTATTTTTTATA TATATATATATTTTTATTTTTT ATATATATATATAGTATATAC SEQ ID NO: 108 GTATATACTTTTTATTTTTGATA TATATATATATTTTTATTTTTT ATATATATATATCGTATATAC SEQ ID NO: 109 GTATATACTTTTTATTTTTGATA TATGTATATATTTTTATTTTTT ATATACATATATCGTATATAC SEQ ID NO: 110 GGATATACTTTTTATTTTTTATA TATATATATATTTTTATTTTTT ATATATATATATAGTATATCC SEQ ID NO: 111 GGATATACTTTTTATTTTTGATA TATATATATATTTTTATTTTTT ATATATATATATCGTATATCC SEQ ID NO: 112 GGATATACTTTTTATTTTTGATA TATGTATATATTTTTATTTTTT ATATACATATATCGTATATCC SEQ ID NO: 113 GGTGATACTTTTTATTTTTTATA TATATATATATTTTTATTTTTT ATATATATATATAGTATCACC SEQ ID NO: 114 GGTGATACTTTTTATTTTTGATA TATATATATATTTTTATTTTTT ATATATATATATCGTATCACC SEQ ID NO: 115 GGTGATACTTTTTATTTTTGATA TATGTATATATTTTTATTTTTT ATATACATATATCGTATCACC SEQ ID NO: 116 GGTGATCCTTTTTATTTTTTATA TATATATATATTTTTATTTTTT ATATATATATATAGGATCACC SEQ ID NO: 117 GGTGATCCTTTTTATTTTTGATA TATATATATATTTTTATTTTTT ATATATATATATCGGATCACC SEQ ID NO: 118 GGTGATCCTTTTTATTTTTGATA TATGTATATATTTTTATTTTTT ATATACATATATCGGATCACC SEQ ID NO: 119 GATATATCACTTTTTATTTTTTA TATATATATATATTTTTATTTT TTATATATATATATAGTGATATA TC SEQ ID NO: 120 GTATATACATTTTTTATTTTTGA TATATATATATATTTTTATTTT TTATATATATATATCATGTATAT AC SEQ ID NO: 121 GTATATACATTTTTTATTTTTGA TATATGTATATATTTTTATTTT TTATATACATATATCATGTATAT AC SEQ ID NO: 122 GGATATACACTTTTTATTTTTTA TATATATATATATTTTTATTTT TTATATATATATATAGTGTATAT CC SEQ ID NO: 123 GGATATACATTTTTTATTTTTGA TATATATATATATTTTTATTTT TTATATATATATATCATGTATAT CC SEQ ID NO: 124 GGATATACATTTTTTATTTTTGA TATATGTATATATTTTTATTTT TTATATACATATATCATGTATAT CC SEQ ID NO: 125 GGGTATATACTTTTTATTTTTTA TATATATATATATTTTTATTTT TTATATATATATATAGTATATAC CC SEQ ID NO: 126 GGATATACACTTTTTATTTTTGA TATATATATATATTTTTATTTT TTATATATATATATCGTGTATAT CC SEQ ID NO: 127 GGATATACACTTTTTATTTTTGA TATATGTATATATTTTTATTTT TTATATACATATATCGTGTATAT CC SEQ ID NO: 128 GGGTATATACTTTTTATTTTTGA TATATATATATATTTTTATTTT TTATATATATATATCGTATATAC CC SEQ ID NO: 129 GGGTATATACTTTTTATTTTTGA TATATGTATATATTTTTATTTT TTATATACATATATCGTATATAC CC SEQ ID NO: 130 GATATATCACTTTTTATTTTTTA TATATATATATTTTTTATTTTT ATATATATATATAGTGATATATC SEQ ID NO: 131 GTATATACATTTTTTATTTTTGA TATATATATATTTTTTATTTTT ATATATATATATCATGTATATAC SEQ ID NO: 132 GTATATACATTTTTTATTTTTGA TATATGTATATTTTTTATTTTT ATATACATATATCATGTATATAC SEQ ID NO: 133 GGATATACACTTTTTATTTTTTA TATATATATATTTTTTATTTTT ATATATATATATAGTGTATATCC SEQ ID NO: 134 GGATATACATTTTTTATTTTTGA TATATATATATTTTTTATTTTT ATATATATATATCATGTATATCC SEQ ID NO: 135 GGATATACATTTTTTATTTTTGA TATATGTATATTTTTTATTTTT ATATACATATATCATGTATATCC SEQ ID NO: 136 GGGTATATACTTTTTATTTTTTA TATATATATATTTTTTATTTTT ATATATATATATAGTATATACCC SEQ ID NO: 137 GGATATACACTTTTTATTTTTGA TATATATATATTTTTTATTTTT ATATATATATATCGTGTATATCC SEQ ID NO: 138 GGATATACACTTTTTATTTTTGA TATATGTATATTTTTTATTTTT ATATACATATATCGTGTATATCC SEQ ID NO: 139 GGGTATATACTTTTTATTTTTGA TATATATATATTTTTTATTTTT ATATATATATATCGTATATACCC SEQ ID NO: 140 GGGTATATACTTTTTATTTTTGA TATATGTATATTTTTTATTTTT ATATACATATATCGTATATACCC SEQ ID NO: 141 GATATATCACTTTTTATTTTTTA TATATATATATTTTTATTTTTT ATATATATATAGTGATATATC SEQ ID NO: 142 GTATATACATTTTTTATTTTTGA TATATATATATTTTTATTTTTT ATATATATATCATGTATATAC SEQ ID NO: 143 GTATATACATTTTTTATTTTTGA TATATGTATATTTTTATTTTTT ATACATATATCATGTATATAC SEQ ID NO: 144 GTATATACATTTTTTATTTTTGA TCATGTATATTTTTTATTTTTA TATACATGATCATGTATATAC SEQ ID NO: 145 GTATATACATTTTTTATTTTTGA TCATATATGTTTTTTATTTTTA CATATATGATCATGTATATAC SEQ ID NO: 146 GGATATACACTTTTTATTTTTTA TATATATATATTTTTATTTTTT ATATATATATAGTGTATATCC SEQ ID NO: 147 GGATATACATTTTTTATTTTTGA TATATATATATTTTTATTTTTT ATATATATATCATGTATATCC SEQ ID NO: 148 GGATATACATTTTTTATTTTTGA TATATGTATATTTTTATTTTTT ATACATATATCATGTATATCC SEQ ID NO: 149 GGATATACATTTTTTATTTTTGA TCATGTATATTTTTTATTTTTA TATACATGATCATGTATATCC SEQ ID NO: 150 GGATATACATTTTTTATTTTTGA TCATATATGTTTTTTATTTTTA CATATATGATCATGTATATCC SEQ ID NO: 151 GGGTATATACTTTTTATTTTTTA TATATATATATTTTTATTTTTT ATATATATATAGTATATACCC SEQ ID NO: 152 GGATATACACTTTTTATTTTTGA TATATATATATTTTTATTTTTT ATATATATATCGTGTATATCC SEQ ID NO: 153 GGATATACACTTTTTATTTTTGA TATATGTATATTTTTATTTTTT ATACATATATCGTGTATATCC SEQ ID NO: 154 GGATATACACTTTTTATTTTTGA TCATGTATATTTTTTATTTTTA TATACATGATCGTGTATATCC SEQ ID NO: 155 GGATATACACTTTTTATTTTTGA TCATATATGTTTTTTATTTTTA CATATATGATCGTGTATATCC SEQ ID NO: 156 GGGTATATACTTTTTATTTTTGA TATATATATATTTTTATTTTTT ATATATATATCGTATATACCC SEQ ID NO: 157 GGGTATATACTTTTTATTTTTGA TATATGTATATTTTTATTTTTT ATACATATATCGTATATACCC SEQ ID NO: 158 GGGTATATACTTTTTATTTTTGA TCATGTATATTTTTTATTTTTA TATACATGATCGTATATACCC SEQ ID NO: 159 GGGTATATACTTTTTATTTTTGA TCATATATGTTTTTTATTTTTA CATATATGATCGTATATACCC SEQ ID NO: 160 GTACATATATTTTTTTATTTTTG ATATATATATTTTTATTTTTTA TATATATCAATATATGTAC SEQ ID NO: 161 GTACATATATTTTTTTATTTTTG ATATATGTATTTTTATTTTTTA CATATATCAATATATGTAC SEQ ID NO: 162 GTACATATATTTTTTTATTTTTG ATCATGTATTTTTTATTTTTAT ACATGATCAATATATGTAC SEQ ID NO: 163 GTACATATATTTTTTTATTTTTG ATCATATATTTTTTATTTTTAT ATATGATCAATATATGTAC SEQ ID NO: 164 GATGTATATACTTTTTATTTTTT ATATATATATTTTTATTTTTTA TATATATAGTATATACATC SEQ ID NO: 165 GGTACATATATTTTTTATTTTTG ATATATATATTTTTATTTTTTA TATATATCATATATGTACC SEQ ID NO: 166 GGTACATATATTTTTTATTTTTG ATATATGTATTTTTATTTTTTA CATATATCATATATGTACC SEQ ID NO: 167 GGTACATATATTTTTTATTTTTG ATCATGTATTTTTTATTTTTAT ACATGATCATATATGTACC SEQ ID NO: 168 GGTACATATATTTTTTATTTTTG ATCATATATTTTTTATTTTTAT ATATGATCATATATGTACC SEQ ID NO: 169 CGATCATATATTTTTTTATTTTT GATATATATATTTTTATTTTTT ATATATATCAATATATGATCG SEQ ID NO: 170 CGATCATATATTTTTTTATTTTT GATATATGTATTTTTATTTTTT ACATATATCAATATATGATCG SEQ ID NO: 171 CGATCATATATTTTTTTATTTTT GATCATGTATTTTTTATTTTTA TACATGATCAATATATGATCG SEQ ID NO: 172 CGATCATATATTTTTTTATTTTT GATCATATATTTTTTATTTTTA TATATGATCAATATATGATCG SEQ ID NO: 173 GATACTTTTTATTTTTTATAAAT ATATATATTTTTTATTTTTATA TATATATATATAGTATC SEQ ID NO: 174 GACACTTTTTATTTTTTATAAAT ATATATATTTTTTATTTTTATA TATATATATATAGTGTC SEQ ID NO: 175 GATACTTTTTATTTTTGATAAAT ATATATATTTTTTATTTTTATA TATATATATATCGTATC SEQ ID NO: 176 GATACTTTTTATTTTTGATAAAT GTATATATTTTTTATTTTTATA TATACATATATCGTATC SEQ ID NO: 177 GATACTTTTTATTTTTGATGATG TATATATATTTTTATTTTTTAT ATATACATGATCGTATC SEQ ID NO: 178 GATACTTTTTATTTTTGATGATA TATGTACTTTTTTATTTTTAGT ACATATATGATCGTATC SEQ ID NO: 179 GGATCTTTTTATTTTTTATAAAT ATATATATTTTTTATTTTTATA TATATATATATAGATCC SEQ ID NO: 180 GACACTTTTTATTTTTGATAAAT ATATATATTTTTTATTTTTATA TATATATATATCGTGTC SEQ ID NO: 181 GACACTTTTTATTTTTGATAAAT GTATATATTTTTTATTTTTATA TATACATATATCGTGTC SEQ ID NO: 182 GACACTTTTTATTTTTGATGATG TATATATATTTTTATTTTTTAT ATATACATGATCGTGTC SEQ ID NO: 183 GACACTTTTTATTTTTGATGATA TATGTACTTTTTTATTTTTAGT ACATATATGATCGTGTC SEQ ID NO: 184 GGATCTTTTTATTTTTGATAAAT ATATATATTTTTTATTTTTATA TATATATATATCGATCC SEQ ID NO: 185 GGATCTTTTTATTTTTGATAAAT GTATATATTTTTTATTTTTATA TATACATATATCGATCC SEQ ID NO: 186 GGATCTTTTTATTTTTGATGATG TATATATATTTTTATTTTTTAT ATATACATGATCGATCC SEQ ID NO: 187 GGATCTTTTTATTTTTGATGATA TATGTACTTTTTTATTTTTAGT ACATATATGATCGATCC SEQ ID NO: 188 GCGTCTTTTTATTTTTTATAAAT ATATATATTTTTTATTTTTATA TATATATATATAGACGC SEQ ID NO: 189 GCGTCTTTTTATTTTTGATAAAT ATATATATTTTTTATTTTTATA TATATATATATCGACGC SEQ ID NO: 190 GCGTCTTTTTATTTTTGATAAAT GTATATATTTTTTATTTTTATA TATACATATATCGACGC SEQ ID NO: 191 GCGTCTTTTTATTTTTGATGATG TATATATATTTTTATTTTTTAT ATATACATGATCGACGC SEQ ID NO: 192 GCGTCTTTTTATTTTTGATGATA TATGTACTTTTTTATTTTTAGT ACATATATGATCGACGC SEQ ID NO: 193 GTATACTTTTTATTTTTGATAAA TATATATATTTTTATTTTTTAT ATATATATATCGTATAC SEQ ID NO: 194 GTATACTTTTTATTTTTGATAAA TGTATATATTTTTATTTTTTAT ATACATATATCGTATAC SEQ ID NO: 195 GTATACTTTTTATTTTTGATGAT GTATATATTTTTTATTTTTATA TATACATGATCGTATAC SEQ ID NO: 196 GTATACTTTTTATTTTTGATGAT ATATGTACTTTTTATTTTTGTA CATATATGATCGTATAC SEQ ID NO: 197 GTGATCTTTTTATTTTTGATAAA TATATATATTTTTATTTTTTAT ATATATATATCGATCAC SEQ ID NO: 198 GTGATCTTTTTATTTTTGATAAA TGTATATATTTTTATTTTTTAT ATACATATATCGATCAC SEQ ID NO: 199 GTGATCTTTTTATTTTTGATGAT GTATATATTTTTTATTTTTATA TATACATGATCGATCAC SEQ ID NO: 200 GTGATCTTTTTATTTTTGATGAT ATATGTACTTTTTATTTTTGTA CATATATGATCGATCAC SEQ ID NO: 201 GGATACTTTTTATTTTTGATAAA TATATATATTTTTATTTTTTAT ATATATATATCGTATCC SEQ ID NO: 202 GGATACTTTTTATTTTTGATAAA TGTATATATTTTTATTTTTTAT ATACATATATCGTATCC SEQ ID NO: 203 GGATACTTTTTATTTTTGATGAT GTATATATTTTTTATTTTTATA TATACATGATCGTATCC SEQ ID NO: 204 GGATACTTTTTATTTTTGATGAT ATATGTACTTTTTATTTTTGTA CATATATGATCGTATCC SEQ ID NO: 205 GCGATCTTTTTATTTTTGATAAA TATATATATTTTTATTTTTTAT ATATATATATCGATCGC SEQ ID NO: 206 GCGATCTTTTTATTTTTGATAAA TGTATATATTTTTATTTTTTAT ATACATATATCGATCGC SEQ ID NO: 207 GCGATCTTTTTATTTTTGATGAT GTATATATTTTTTATTTTTATA TATACATGATCGATCGC SEQ ID NO: 208 GCGATCTTTTTATTTTTGATGAT ATATGTACTTTTTATTTTTGTA CATATATGATCGATCGC SEQ ID NO: 209 GATATACTTTTTATTTTTTATAA ATATATATTTTTTATTTTTATA TATATATATAGTATATC SEQ ID NO: 210 GATATATTTTTTATTTTTGATAA ATGTATATTTTTTATTTTTATA TACATATATCATATATC SEQ ID NO: 211 GATATATTTTTTATTTTTGATGA TGTATATATTTTTATTTTTTAT ATACATGATCATATATC SEQ ID NO: 212 GATATATTTTTTATTTTTGATGA TATATGTATTTTTATTTTTTAC ATATATGATCATATATC SEQ ID NO: 213 GTGATACTTTTTATTTTTTATAA ATATATATTTTTTATTTTTATA TATATATATAGTATCAC SEQ ID NO: 214 GATATACTTTTTATTTTTGATAA ATATATATTTTTTATTTTTATA TATATATATCGTATATC SEQ ID NO: 215 GATATACTTTTTATTTTTGATGA TGTATATATTTTTATTTTTTAT ATACATGATCGTATATC SEQ ID NO: 216 GATATACTTTTTATTTTTGATGA TATATGTATTTTTATTTTTTAC ATATATGATCGTATATC SEQ ID NO: 217 GGTATACTTTTTATTTTTTATAA ATATATATTTTTTATTTTTATA TATATATATAGTATACC SEQ ID NO: 218 GTGATACTTTTTATTTTTGATAA ATATATATTTTTTATTTTTATA TATATATATCGTATCAC SEQ ID NO: 219 GTGATACTTTTTATTTTTGATAA ATGTATATTTTTTATTTTTATA TACATATATCGTATCAC SEQ ID NO: 220 GTGATACTTTTTATTTTTGATGA TGTATATATTTTTATTTTTTAT ATACATGATCGTATCAC SEQ ID NO: 221 GTGATACTTTTTATTTTTGATGA TATATGTATTTTTATTTTTTAC ATATATGATCGTATCAC SEQ ID NO: 222 GGTATACTTTTTATTTTTGATAA ATATATATTTTTTATTTTTATA TATATATATCGTATACC SEQ ID NO: 223 GGTATACTTTTTATTTTTGATAA ATGTATATTTTTTATTTTTATA TACATATATCGTATACC SEQ ID NO: 224 GGTATACTTTTTATTTTTGATGA TGTATATATTTTTATTTTTTAT ATACATGATCGTATACC SEQ ID NO: 225 GGTATACTTTTTATTTTTGATGA TATATGTATTTTTATTTTTTAC ATATATGATCGTATACC SEQ ID NO: 226 GGTGTACTTTTTATTTTTTATAA ATATATATTTTTTATTTTTATA TATATATATAGTACACC SEQ ID NO: 227 GGTGTACTTTTTATTTTTGATAA ATATATATTTTTTATTTTTATA TATATATATCGTACACC SEQ ID NO: 228 GGTGTACTTTTTATTTTTGATAA ATGTATATTTTTTATTTTTATA TACATATATCGTACACC SEQ ID NO: 229 GGTGTACTTTTTATTTTTGATGA TGTATATATTTTTATTTTTTAT ATACATGATCGTACACC SEQ ID NO: 230 GGTGTACTTTTTATTTTTGATGA TATATGTATTTTTATTTTTTAC ATATATGATCGTACACC SEQ ID NO: 231 GTATATACTTTTTATTTTTGATA AATATATATTTTTATTTTTTAT ATATATATCGTATATAC SEQ ID NO: 232 GTATATACTTTTTATTTTTGATA AATGTATATTTTTATTTTTTAT ACATATATCGTATATAC SEQ ID NO: 233 GTATATACTTTTTATTTTTGATG ATGTATATTTTTTATTTTTATA TACATGATCGTATATAC SEQ ID NO: 234 GTATATACTTTTTATTTTTGATG ATATATGTTTTTTATTTTTACA TATATGATCGTATATAC SEQ ID NO: 235 GGATATACTTTTTATTTTTGATA AATATATATTTTTATTTTTTAT ATATATATCGTATATCC SEQ ID NO: 236 GGATATACTTTTTATTTTTGATA AATGTATATTTTTATTTTTTAT ACATATATCGTATATCC SEQ ID NO: 237 GGATATACTTTTTATTTTTGATG ATGTATATTTTTTATTTTTATA TACATGATCGTATATCC SEQ ID NO: 238 GGATATACTTTTTATTTTTGATG ATATATGTTTTTTATTTTTACA TATATGATCGTATATCC SEQ ID NO: 239 GGTGATACTTTTTATTTTTGATA AATATATATTTTTATTTTTTAT ATATATATCGTATCACC SEQ ID NO: 240 GGTGATACTTTTTATTTTTGATA AATGTATATTTTTATTTTTTAT ACATATATCGTATCACC SEQ ID NO: 241 GGTGATACTTTTTATTTTTGATG ATGTATATTTTTTATTTTTATA TACATGATCGTATCACC SEQ ID NO: 242 GGTGATACTTTTTATTTTTGATG ATATATGTTTTTTATTTTTACA TATATGATCGTATCACC SEQ ID NO: 243 GGTGATCCTTTTTATTTTTGATA AATATATATTTTTATTTTTTAT ATATATATCGGATCACC SEQ ID NO: 244 GGTGATCCTTTTTATTTTTGATA AATGTATATTTTTATTTTTTAT ACATATATCGGATCACC SEQ ID NO: 245 GGTGATCCTTTTTATTTTTGATG ATGTATATTTTTTATTTTTATA TACATGATCGGATCACC SEQ ID NO: 246 GGTGATCCTTTTTATTTTTGATG ATATATGTTTTTTATTTTTACA TATATGATCGGATCACC SEQ ID NO: 247 GTATATACATTTTTTATTTTTGA TAAATATATTTTTATTTTTTAT ATATATCATGTATATAC SEQ ID NO: 248 GTATATACATTTTTTATTTTTGA TAAATGTATTTTTATTTTTTAC ATATATCATGTATATAC SEQ ID NO: 249 GTATATACATTTTTTATTTTTGA TGATGTATTTTTTATTTTTATA CATGATCATGTATATAC SEQ ID NO: 250 GTATATACATTTTTTATTTTTGA TGATATATTTTTTATTTTTATA TATGATCATGTATATAC SEQ ID NO: 251 GGATATACATTTTTTATTTTTGA TAAATATATTTTTATTTTTTAT ATATATCATGTATATCC SEQ ID NO: 252 GGATATACATTTTTTATTTTTGA TAAATGTATTTTTATTTTTTAC ATATATCATGTATATCC SEQ ID NO: 253 GGATATACATTTTTTATTTTTGA TGATGTATTTTTTATTTTTATA CATGATCATGTATATCC SEQ ID NO: 254 GGATATACATTTTTTATTTTTGA TGATATATTTTTTATTTTTATA TATGATCATGTATATCC SEQ ID NO: 255 GGATATACACTTTTTATTTTTGA TAAATATATTTTTATTTTTTAT ATATATCGTGTATATCC SEQ ID NO: 256 GGATATACACTTTTTATTTTTGA TAAATGTATTTTTATTTTTTAC ATATATCGTGTATATCC SEQ ID NO: 257 GGATATACACTTTTTATTTTTGA TGATGTATTTTTTATTTTTATA CATGATCGTGTATATCC SEQ ID NO: 258 GGATATACACTTTTTATTTTTGA TGATATATTTTTTATTTTTATA TATGATCGTGTATATCC SEQ ID NO: 259 GGGTATATACTTTTTATTTTTGA TAAATATATTTTTATTTTTTAT ATATATCGTATATACCC SEQ ID NO: 260 GGGTATATACTTTTTATTTTTGA TAAATGTATTTTTATTTTTTAC ATATATCGTATATACCC SEQ ID NO: 261 GGGTATATACTTTTTATTTTTGA TGATGTATTTTTTATTTTTATA CATGATCGTATATACCC SEQ ID NO: 262 GGGTATATACTTTTTATTTTTGA TGATATATTTTTTATTTTTATA TATGATCGTATATACCC SEQ ID NO: 263 GGATGTACACTTTTTATTTTTGA TAAATATATTTTTATTTTTTAT ATATATCGTGTACATCC SEQ ID NO: 264 GGATGTACACTTTTTATTTTTGA TAAATGTATTTTTATTTTTTAC ATATATCGTGTACATCC SEQ ID NO: 265 GGATGTACACTTTTTATTTTTGA TGATGTATTTTTTATTTTTATA CATGATCGTGTACATCC SEQ ID NO: 266 GGATGTACACTTTTTATTTTTGA TGATATATTTTTTATTTTTATA TATGATCGTGTACATCC SEQ ID NO: 267 GTATATACTTTTTATTTTTTATA AATATATATATTTTTTATTTTT ATATATATATATATAGTATATAC SEQ ID NO: 268 GTATATACTTTTTATTTTTGATA AATATATATATTTTTTATTTTT ATATATATATATATCGTATATAC SEQ ID NO: 269 GTATATACTTTTTATTTTTGATA AATGTATATATTTTTTATTTTT ATATATACATATATCGTATATAC SEQ ID NO: 270 GTATATACTTTTTATTTTTGATG ATGTATATATATTTTTATTTTT TATATATACATGATCGTATATAC SEQ ID NO: 271 GTATATACTTTTTATTTTTGATG ATATATGTACTTTTTTATTTTT AGTACATATATGATCGTATATAC SEQ ID NO: 272 GGATATACTTTTTATTTTTTATA AATATATATATTTTTTATTTTT ATATATATATATATAGTATATCC SEQ ID NO: 273 GGATATACTTTTTATTTTTGATA AATATATATATTTTTTATTTTT ATATATATATATATCGTATATCC SEQ ID NO: 274 GGATATACTTTTTATTTTTGATA AATGTATATATTTTTTATTTTT ATATATACATATATCGTATATCC SEQ ID NO: 275 GGATATACTTTTTATTTTTGATG ATGTATATATATTTTTATTTTT TATATATACATGATCGTATATCC SEQ ID NO: 276 GGATATACTTTTTATTTTTGATG ATATATGTACTTTTTTATTTTT AGTACATATATGATCGTATATCC SEQ ID NO: 277 GGTGATACTTTTTATTTTTTATA AATATATATATTTTTTATTTTT ATATATATATATATAGTATCACC SEQ ID NO: 278 GGTGATACTTTTTATTTTTGATA AATATATATATTTTTTATTTTT ATATATATATATATCGTATCACC SEQ ID NO: 279 GGTGATACTTTTTATTTTTGATA AATGTATATATTTTTTATTTTT ATATATACATATATCGTATCACC SEQ ID NO: 280 GGTGATACTTTTTATTTTTGATG ATGTATATATATTTTTATTTTT TATATATACATGATCGTATCACC SEQ ID NO: 281 GGTGATACTTTTTATTTTTGATG ATATATGTACTTTTTTATTTTT AGTACATATATGATCGTATCACC SEQ ID NO: 282 GGTGATCCTTTTTATTTTTTATA AATATATATATTTTTTATTTTT ATATATATATATATAGGATCACC SEQ ID NO: 283 GGTGATCCTTTTTATTTTTGATA AATATATATATTTTTTATTTTT ATATATATATATATCGGATCACC SEQ ID NO: 284 GGTGATCCTTTTTATTTTTGATA AATGTATATATTTTTTATTTTT ATATATACATATATCGGATCACC SEQ ID NO: 285 GGTGATCCTTTTTATTTTTGATG ATGTATATATATTTTTATTTTT TATATATACATGATCGGATCACC SEQ ID NO: 286 GGTGATCCTTTTTATTTTTGATG ATATATGTACTTTTTTATTTTT AGTACATATATGATCGGATCACC SEQ ID NO: 287 GATATATCACTTTTTATTTTTTA TAAATATATATATTTTTTATTT TTATATATATATATATAGTGATA TATC SEQ ID NO: 288 GTATATACATTTTTTATTTTTGA TAAATATATATATTTTTTATTT TTATATATATATATATCATGTAT ATAC SEQ ID NO: 289 GTATATACATTTTTTATTTTTGA TAAATGTATATATTTTTTATTT TTATATATACATATATCATGTAT ATAC SEQ ID NO: 290 GTATATACATTTTTTATTTTTGA TGATGTATATATATTTTTATTT TTTATATATACATGATCATGTAT ATAC SEQ ID NO: 291 GTATATACATTTTTTATTTTTGA TGATATATGTACTTTTTTATTT TTAGTACATATATGATCATGTAT ATAC SEQ ID NO: 292 GGATATACACTTTTTATTTTTTA TAAATATATATATTTTTTATTT TTATATATATATATATAGTGTAT ATCC SEQ ID NO: 293 GGATATACATTTTTTATTTTTGA TAAATATATATATTTTTTATTT TTATATATATATATATCATGTAT ATCC SEQ ID NO: 294 GGATATACATTTTTTATTTTTGA TAAATGTATATATTTTTTATTT TTATATATACATATATCATGTAT ATCC SEQ ID NO: 295 GGATATACATTTTTTATTTTTGA TGATGTATATATATTTTTATTT TTTATATATACATGATCATGTAT ATCC SEQ ID NO: 296 GGATATACATTTTTTATTTTTGA TGATATATGTACTTTTTTATTT TTAGTACATATATGATCATGTAT ATCC SEQ ID NO: 297 GGGTATATACTTTTTATTTTTTA TAAATATATATATTTTTTATTT TTATATATATATATATAGTATAT ACCC SEQ ID NO: 298 GGATATACACTTTTTATTTTTGA TAAATATATATATTTTTTATT TTTATATATATATATATCGTGTA TATCC SEQ ID NO: 299 GGATATACACTTTTTATTTTTGA TAAATGTATATATTTTTTATT TTTATATATACATATATCGTGTA TATCC SEQ ID NO: 300 GGATATACACTTTTTATTTTTGA TGATGTATATATATTTTTATT TTTTATATATACATGATCGTGTA TATCC SEQ ID NO: 301 GGATATACACTTTTTATTTTTGA TGATATATGTACTTTTTTATTT TTAGTACATATATGATCGTGTAT ATCC SEQ ID NO: 302 GGGTATATACTTTTTATTTTTGA TAAATATATATATTTTTTATTT TTATATATATATATATCGTATAT ACCC SEQ ID NO: 303 GGGTATATACTTTTTATTTTTGA TAAATGTATATATTTTTTATTT TTATATATACATATATCGTATAT ACCC SEQ ID NO: 304 GGGTATATACTTTTTATTTTTGA TGATGTATATATATTTTTATTT TTTATATATACATGATCGTATAT ACCC SEQ ID NO: 305 GGGTATATACTTTTTATTTTTGA TGATATATGTACTTTTTTATTT TTAGTACATATATGATCGTATAT ACCC SEQ ID NO: 306 GTATATACTTTTTATTTTTGATA AATATATATATTTTTATTTTTT ATATATATATATCGTATATAC SEQ ID NO: 307 GTATATACTTTTTATTTTTGATA AATGTATATATTTTTATTTTTT ATATACATATATCGTATATAC SEQ ID NO: 308 GTATATACTTTTTATTTTTGATG ATGTATATATTTTTTATTTTTA TATATACATGATCGTATATAC SEQ ID NO: 309 GTATATACTTTTTATTTTTGATG ATATATGTACTTTTTATTTTTG TACATATATGATCGTATATAC SEQ ID NO: 310 GGATATACTTTTTATTTTTGATA AATATATATATTTTTATTTTTT ATATATATATATCGTATATCC SEQ ID NO: 311 GGATATACTTTTTATTTTTGATA AATGTATATATTTTTATTTTTT ATATACATATATCGTATATCC SEQ ID NO: 312 GGATATACTTTTTATTTTTGATG ATGTATATATTTTTTATTTTTA TATATACATGATCGTATATCC SEQ ID NO: 313 GGATATACTTTTTATTTTTGATG ATATATGTACTTTTTATTTTTG TACATATATGATCGTATATCC SEQ ID NO: 314 GGTGATACTTTTTATTTTTGATA AATATATATATTTTTATTTTTT ATATATATATATCGTATCACC SEQ ID NO: 315 GGTGATACTTTTTATTTTTGATA AATGTATATATTTTTATTTTTT ATATACATATATCGTATCACC SEQ ID NO: 316 GGTGATACTTTTTATTTTTGATG ATGTATATATTTTTTATTTTTA TATATACATGATCGTATCACC SEQ ID NO: 317 GGTGATACTTTTTATTTTTGATG ATATATGTACTTTTTATTTTTG TACATATATGATCGTATCACC SEQ ID NO: 318 GGTGATCCTTTTTATTTTTGATA AATATATATATTTTTATTTTTT ATATATATATATCGGATCACC SEQ ID NO: 319 GGTGATCCTTTTTATTTTTGATA AATGTATATATTTTTATTTTTT ATATACATATATCGGATCACC SEQ ID NO: 320 GGTGATCCTTTTTATTTTTGATG ATGTATATATTTTTTATTTTTA TATATACATGATCGGATCACC SEQ ID NO: 321 GGTGATCCTTTTTATTTTTGATG ATATATGTACTTTTTATTTTTG TACATATATGATCGGATCACC SEQ ID NO: 322 GTATATACATTTTTTATTTTTGA TAAATATATATATTTTTATTTT TTATATATATATATCATGTATAT AC SEQ ID NO: 323 GTATATACATTTTTTATTTTTGA TGATGTATATATTTTTTATTTT TATATATACATGATCATGTATAT AC SEQ ID NO: 324 GTATATACATTTTTTATTTTTGA TGATATATGTACTTTTTATTTT TGTACATATATGATCATGTATAT AC SEQ ID NO: 325 GGATATACATTTTTTATTTTTGA TAAATATATATATTTTTATTTT TTATATATATATATCATGTATAT CC SEQ ID NO: 326 GGATATACATTTTTTATTTTTGA TAAATGTATATATTTTTATTTT TTATATACATATATCATGTATAT CC SEQ ID NO: 327 GGATATACATTTTTTATTTTTGA TGATGTATATATTTTTTATTTT TATATATACATGATCATGTATAT CC SEQ ID NO: 328 GGATATACATTTTTTATTTTTGA TGATATATGTACTTTTTATTTT TGTACATATATGATCATGTATAT CC SEQ ID NO: 329 GGATATACACTTTTTATTTTTGA TAAATATATATATTTTTATTT TTTATATATATATATCGTGTATA TCC SEQ ID NO: 330 GGATATACACTTTTTATTTTTGA TAAATGTATATATTTTTATTT TTTATATACATATATCGTGTATA TCC SEQ ID NO: 331 GGATATACACTTTTTATTTTTGA TGATGTATATATTTTTTATTTT TATATATACATGATCGTGTATAT CC SEQ ID NO: 332 GGATATACACTTTTTATTTTTGA TGATATATGTACTTTTTATTTT TGTACATATATGATCGTGTATAT CC SEQ ID NO: 333 GGGTATATACTTTTTATTTTTGA TAAATATATATATTTTTATTTT TTATATATATATATCGTATATAC CC SEQ ID NO: 334 GGGTATATACTTTTTATTTTTGA TAAATGTATATATTTTTATTTT TTATATACATATATCGTATATAC CC SEQ ID NO: 335 GGGTATATACTTTTTATTTTTGA TGATGTATATATTTTTTATTTT TATATATACATGATCGTATATAC CC SEQ ID NO: 336 GGGTATATACTTTTTATTTTTGA TGATATATGTACTTTTTATTTT TGTACATATATGATCGTATATAC CC SEQ ID NO: 337 GATATATCACTTTTTATTTTTTA TAAATATATATTTTTTATTTTT ATATATATATATAGTGATATATC SEQ ID NO: 338 GTATATACATTTTTTATTTTTGA TAAATATATATTTTTTATTTTT ATATATATATATCATGTATATAC SEQ ID NO: 339 GTATATACATTTTTTATTTTTGA TGATGTATATATTTTTATTTTT TATATACATGATCATGTATATAC SEQ ID NO: 340 GTATATACATTTTTTATTTTTGA TGATATATGTATTTTTATTTTT TACATATATGATCATGTATATAC SEQ ID NO: 341 GGATATACACTTTTTATTTTTTA TAAATATATATTTTTTATTTTT ATATATATATATAGTGTATATCC SEQ ID NO: 342 GGATATACATTTTTTATTTTTGA TAAATATATATTTTTTATTTTT ATATATATATATCATGTATATCC SEQ ID NO: 343 GGATATACATTTTTTATTTTTGA TAAATGTATATTTTTTATTTTT ATATACATATATCATGTATATCC SEQ ID NO: 344 GGATATACATTTTTTATTTTTGA TGATGTATATATTTTTATTTTT TATATACATGATCATGTATATCC SEQ ID NO: 345 GGATATACATTTTTTATTTTTGA TGATATATGTATTTTTATTTTT TACATATATGATCATGTATATCC SEQ ID NO: 346 GGGTATATACTTTTTATTTTTTA TAAATATATATTTTTTATTTTT ATATATATATATAGTATATACCC SEQ ID NO: 347 GGATATACACTTTTTATTTTTGA TAAATATATATTTTTTATTTTT ATATATATATATCGTGTATATCC SEQ ID NO: 348 GGATATACACTTTTTATTTTTGA TAAATGTATATTTTTTATTTTT ATATACATATATCGTGTATATCC SEQ ID NO: 349 GGATATACACTTTTTATTTTTGA TGATGTATATATTTTTATTTTT TATATACATGATCGTGTATATCC SEQ ID NO: 350 GGATATACACTTTTTATTTTTGA TGATATATGTATTTTTATTTTT TACATATATGATCGTGTATATCC SEQ ID NO: 351 GGGTATATACTTTTTATTTTTGA TAAATATATATTTTTTATTTTT ATATATATATATCGTATATACCC SEQ ID NO: 352 GGGTATATACTTTTTATTTTTGA TAAATGTATATTTTTTATTTTT ATATACATATATCGTATATACCC SEQ ID NO: 353 GGGTATATACTTTTTATTTTTGA TGATGTATATATTTTTATTTTT TATATACATGATCGTATATACCC SEQ ID NO: 354 GGGTATATACTTTTTATTTTTGA TGATATATGTATTTTTATTTTT TACATATATGATCGTATATACCC SEQ ID NO: 355 GTATATACATTTTTTATTTTTGA TAAATATATATTTTTATTTTTT ATATATATATCATGTATATAC SEQ ID NO: 356 GTATATACATTTTTTATTTTTGA TGATGTATATTTTTTATTTTTA TATACATGATCATGTATATAC SEQ ID NO: 357 GTATATACATTTTTTATTTTTGA TGATATATGTTTTTTATTTTTA CATATATGATCATGTATATAC SEQ ID NO: 358 GGATATACATTTTTTATTTTTGA TAAATATATATTTTTATTTTTT ATATATATATCATGTATATCC SEQ ID NO: 359 GGATATACATTTTTTATTTTTGA TAAATGTATATTTTTATTTTTT ATACATATATCATGTATATCC SEQ ID NO: 360 GGATATACATTTTTTATTTTTGA TGATGTATATTTTTTATTTTTA TATACATGATCATGTATATCC SEQ ID NO: 361 GGATATACATTTTTTATTTTTGA TGATATATGTTTTTTATTTTTA CATATATGATCATGTATATCC SEQ ID NO: 362 GGATATACACTTTTTATTTTTGA TAAATATATATTTTTATTTTTT ATATATATATCGTGTATATCC SEQ ID NO: 363 GGATATACACTTTTTATTTTTGA TAAATGTATATTTTTATTTTTT ATACATATATCGTGTATATCC SEQ ID NO: 364 GGATATACACTTTTTATTTTTGA TGATGTATATTTTTTATTTTTA TATACATGATCGTGTATATCC SEQ ID NO: 365 GGATATACACTTTTTATTTTTGA TGATATATGTTTTTTATTTTTA CATATATGATCGTGTATATCC SEQ ID NO: 366 GGGTATATACTTTTTATTTTTGA TAAATATATATTTTTATTTTTT ATATATATATCGTATATACCC SEQ ID NO: 367 GGGTATATACTTTTTATTTTTGA TAAATGTATATTTTTATTTTTT ATACATATATCGTATATACCC SEQ ID NO: 368 GGGTATATACTTTTTATTTTTGA TGATGTATATTTTTTATTTTTA TATACATGATCGTATATACCC SEQ ID NO: 369 GGGTATATACTTTTTATTTTTGA TGATATATGTTTTTTATTTTTA CATATATGATCGTATATACCC SEQ ID NO: 370 GTACATATATTTTTTTATTTTTG ATAAATATATTTTTATTTTTTA TATATATCAATATATGTAC SEQ ID NO: 371 GTACATATATTTTTTTATTTTTG ATAAATGTATTTTTATTTTTTA CATATATCAATATATGTAC SEQ ID NO: 372 GTACATATATTTTTTTATTTTTG ATGATGTATTTTTTATTTTTAT ACATGATCAATATATGTAC SEQ ID NO: 373 GTACATATATTTTTTTATTTTTG ATGATATATTTTTTATTTTTAT ATATGATCAATATATGTAC SEQ ID NO: 374 GGTACATATATTTTTTATTTTTG ATAAATATATTTTTATTTTTTA TATATATCATATATGTACC SEQ ID NO: 375 GGTACATATATTTTTTATTTTTG ATAAATGTATTTTTATTTTTTA CATATATCATATATGTACC SEQ ID NO: 376 GGTACATATATTTTTTATTTTTG ATGATGTATTTTTTATTTTTAT ACATGATCATATATGTACC SEQ ID NO: 377 GGTACATATATTTTTTATTTTTG ATGATATATTTTTTATTTTTAT ATATGATCATATATGTACC SEQ ID NO: 378 CGATCATATATTTTTTTATTTTT GATAAATATATTTTTATTTTTT ATATATATCAATATATGATCG SEQ ID NO: 379 CGATCATATATTTTTTTATTTTT GATAAATGTATTTTTATTTTTT ACATATATCAATATATGATCG SEQ ID NO: 380 CGATCATATATTTTTTTATTTTT GATGATGTATTTTTTATTTTTA TACATGATCAATATATGATCG SEQ ID NO: 381 CGATCATATATTTTTTTATTTTT GATGATATATTTTTTATTTTTA TATATGATCAATATATGATCG SEQ ID NO: 382 GTATATACTTTTTATTTTTGATG ATGTAAATATATTTTTATTTTT TATATATACATGATCGTATATAC SEQ ID NO: 383 GTATATACTTTTTATTTTTGATG ATATAAGTACTTTTTTATTTTT AGTACATATATGATCGTATATAC SEQ ID NO: 384 GGATATACTTTTTATTTTTGATG ATGTAAATATATTTTTATTTTT TATATATACATGATCGTATATCC SEQ ID NO: 385 GGATATACTTTTTATTTTTGATG ATATAAGTACTTTTTTATTTTT AGTACATATATGATCGTATATCC SEQ ID NO: 386 GGTGATACTTTTTATTTTTGATG ATGTAAATATATTTTTATTTTT TATATATACATGATCGTATCACC SEQ ID NO: 387 GGTGATACTTTTTATTTTTGATG ATATAAGTACTTTTTTATTTTT AGTACATATATGATCGTATCACC SEQ ID NO: 388 GGTGATCCTTTTTATTTTTGATG ATGTAAATATATTTTTATTTTT TATATATACATGATCGGATCACC SEQ ID NO: 389 GGTGATCCTTTTTATTTTTGATG ATATAAGTACTTTTTTATTTTT AGTACATATATGATCGGATCACC SEQ ID NO: 390 GTATATACATTTTTTATTTTTGA TGATGTAAATATATTTTTATTT TTTATATATACATGATCATGTAT ATAC SEQ ID NO: 391 GTATATACATTTTTTATTTTTGA TGATATAAGTACTTTTTTATTT TTAGTACATATATGATCATGTAT ATAC SEQ ID NO: 392 GGATATACATTTTTTATTTTTGA TAAATGTAAATATTTTTTATT TTTATATATACATATATCATGTA TATCC SEQ ID NO: 393 GGATATACATTTTTTATTTTTGA TGATGTAAATATATTTTTATT TTTTATATATACATGATCATGTA TATCC SEQ ID NO: 394 GGATATACATTTTTTATTTTTGA TGATATAAGTACTTTTTTATT TTTAGTACATATATGATCATGTA TATCC SEQ ID NO: 395 GGATATACACTTTTTATTTTTGA TGATGTAAATATATTTTTATT TTTTATATATACATGATCGTGTA TATCC SEQ ID NO: 396 GGATATACACTTTTTATTTTTGA TGATATAAGTACTTTTTTATT TTTAGTACATATATGATCGTGTA TATCC SEQ ID NO: 397 GGGTATATACTTTTTATTTTTGA TGATGTAAATATATTTTTATT TTTTATATATACATGATCGTATA TACCC SEQ ID NO: 398 GGGTATATACTTTTTATTTTTGA TGATATAAGTACTTTTTTATT TTTAGTACATATATGATCGTATA TACCC SEQ ID NO: 399 GTATATACTTTTTATTTTTGATG ATGTAAATATTTTTTATTTTTA TATATACATGATCGTATATAC SEQ ID NO: 400 GTATATACTTTTTATTTTTGATG ATATAAGTACTTTTTATTTTTG TACATATATGATCGTATATAC SEQ ID NO: 401 GGATATACTTTTTATTTTTGATG ATGTAAATATTTTTTATTTTTA TATATACATGATCGTATATCC SEQ ID NO: 402 GGATATACTTTTTATTTTTGATG ATATAAGTACTTTTTATTTTT GTACATATATGATCGTATATCC SEQ ID NO: 403 GGTGATACTTTTTATTTTTGATG ATGTAAATATTTTTTATTTTTA TATATACATGATCGTATCACC SEQ ID NO: 404 GGTGATACTTTTTATTTTTGATG ATATAAGTACTTTTTATTTTT GTACATATATGATCGTATCACC SEQ ID NO: 405 GGTGATCCTTTTTATTTTTGATG ATGTAAATATTTTTTATTTTTA TATATACATGATCGGATCACC SEQ ID NO: 406 GGTGATCCTTTTTATTTTTGATG ATATAAGTACTTTTTATTTTTG TACATATATGATCGGATCACC SEQ ID NO: 407 GTATATACATTTTTTATTTTTGA TGATGTAAATATTTTTTATTTT TATATATACATGATCATGTATAT AC SEQ ID NO: 408 GTATATACATTTTTTATTTTTGA TGATATAAGTACTTTTTATTTT TGTACATATATGATCATGTATAT AC SEQ ID NO: 409 GGATATACATTTTTTATTTTTGA TGATGTAAATATTTTTTATTTT TATATATACATGATCATGTATAT CC SEQ ID NO: 410 GGATATACATTTTTTATTTTTGA TGATATAAGTACTTTTTATTT TTGTACATATATGATCATGTATA TCC SEQ ID NO: 411 GGATATACACTTTTTATTTTTGA TGATGTAAATATTTTTTATTT TTATATATACATGATCGTGTATA TCC SEQ ID NO: 412 GGATATACACTTTTTATTTTTGA TGATATAAGTACTTTTTATTT TTGTACATATATGATCGTGTATA TCC SEQ ID NO: 413 GGGTATATACTTTTTATTTTTGA TGATGTAAATATTTTTTATTTT TATATATACATGATCGTATATAC CC SEQ ID NO: 414 GGGTATATACTTTTTATTTTTGA TGATATAAGTACTTTTTATTT TTGTACATATATGATCGTATATA CCC SEQ ID NO: 415 GTATATACATTTTTTATTTTTGA TGATATAAGTATTTTTATTTTT TACATATATGATCATGTATATAC SEQ ID NO: 416 GGATATACATTTTTTATTTTTGA TAAATGAATATTTTTTATTTTT ATATACATATATCATGTATATCC SEQ ID NO: 417 GGATATACATTTTTTATTTTTGA TGATATAAGTATTTTTATTTTT TACATATATGATCATGTATATCC SEQ ID NO: 418 GGATATACACTTTTTATTTTTGA TAAATGAATATTTTTTATTTT TATATACATATATCGTGTATATC C SEQ ID NO: 419 GGATATACACTTTTTATTTTTGA TGATATAAGTATTTTTATTTT TTACATATATGATCGTGTATATC C SEQ ID NO: 420 GGGTATATACTTTTTATTTTTGA TAAATGAATATTTTTTATTTTT ATATACATATATCGTATATACCC SEQ ID NO: 421 GGGTATATACTTTTTATTTTTGA TGATATAAGTATTTTTATTTTT TACATATATGATCGTATATACCC SEQ ID NO: 422 GTATATACATTTTTTATTTTTGA TGATGAATATTTTTTATTTTTA TATACATGATCATGTATATAC SEQ ID NO: 423 GGATATACATTTTTTATTTTTGA TAAATGAATATTTTTATTTTTT ATACATATATCATGTATATCC SEQ ID NO: 424 GGATATACATTTTTTATTTTTGA TGATGAATATTTTTTATTTTTA TATACATGATCATGTATATCC SEQ ID NO: 425 GGATATACATTTTTTATTTTTGA TGATAAATGTTTTTTATTTTTA CATATATGATCATGTATATCC SEQ ID NO: 426 GGATATACACTTTTTATTTTTGA TGATGAATATTTTTTATTTTT ATATACATGATCGTGTATATCC SEQ ID NO: 427 GGGTATATACTTTTTATTTTTGA TGATGAATATTTTTTATTTTTA TATACATGATCGTATATACCC SEQ ID NO: 428 GATACTTTTTATTTTTGATGATG TAAATATATTTTTATTTTTTAT ATATACATGATCGTATC SEQ ID NO: 429 GATACTTTTTATTTTTGATGATA TAAGTACTTTTTTATTTTTAGT ACATATATGATCGTATC SEQ ID NO: 430 GACACTTTTTATTTTTGATGATG TAAATATATTTTTATTTTTTAT ATATACATGATCGTGTC SEQ ID NO: 431 GACACTTTTTATTTTTGATGATA TAAGTACTTTTTTATTTTTAGT ACATATATGATCGTGTC SEQ ID NO: 432 GGATCTTTTTATTTTTGATGATG TAAATATATTTTTATTTTTTAT ATATACATGATCGATCC SEQ ID NO: 433 GGATCTTTTTATTTTTGATGATA TAAGTACTTTTTTATTTTTAGT ACATATATGATCGATCC SEQ ID NO: 434 GCGTCTTTTTATTTTTGATGATG TAAATATATTTTTATTTTTTAT ATATACATGATCGACGC SEQ ID NO: 435 GCGTCTTTTTATTTTTGATGATA TAAGTACTTTTTTATTTTTAGT ACATATATGATCGACGC SEQ ID NO: 436 GTATACTTTTTATTTTTGATGAT GTAAATATTTTTTATTTTTATA TATACATGATCGTATAC SEQ ID NO: 437 GTATACTTTTTATTTTTGATGAT ATAAGTACTTTTTATTTTTGTA CATATATGATCGTATAC SEQ ID NO: 438 GTGATCTTTTTATTTTTGATGAT GTAAATATTTTTTATTTTTATA TATACATGATCGATCAC SEQ ID NO: 439 GTGATCTTTTTATTTTTGATGAT ATAAGTACTTTTTATTTTTGTA CATATATGATCGATCAC SEQ ID NO: 440 GGATACTTTTTATTTTTGATGAT GTAAATATTTTTTATTTTTATA TATACATGATCGTATCC SEQ ID NO: 441 GGATACTTTTTATTTTTGATGAT ATAAGTACTTTTTATTTTTGT ACATATATGATCGTATCC SEQ ID NO: 442 GCGATCTTTTTATTTTTGATGAT GTAAATATTTTTTATTTTTATA TATACATGATCGATCGC SEQ ID NO: 443 GCGATCTTTTTATTTTTGATGAT ATAAGTACTTTTTATTTTTGTA CATATATGATCGATCGC SEQ ID NO: 444 GATATATTTTTTATTTTTGATGA TATAAGTATTTTTATTTTTTAC ATATATGATCATATATC SEQ ID NO: 445 GATATACTTTTTATTTTTGATGA TATAAGTATTTTTATTTTTTAC ATATATGATCGTATATC SEQ ID NO: 446 GTGATACTTTTTATTTTTGATAA ATGAATATTTTTTATTTTTATA TACATATATCGTATCAC SEQ ID NO: 447 GTGATACTTTTTATTTTTGATGA TATAAGTATTTTTATTTTTTAC ATATATGATCGTATCAC SEQ ID NO: 448 GGTATACTTTTTATTTTTGATAA ATGAATATTTTTTATTTTTATA TACATATATCGTATACC SEQ ID NO: 449 GGTATACTTTTTATTTTTGATGA TATAAGTATTTTTATTTTTTAC ATATATGATCGTATACC SEQ ID NO: 450 GGTGTACTTTTTATTTTTGATAA ATGAATATTTTTTATTTTTATA TACATATATCGTACACC SEQ ID NO:451 GGTGTACTTTTTATTTTTGATGA TATAAGTATTTTTATTTTTTAC ATATATGATCGTACACC SEQ ID NO: 452 GTATATACTTTTTATTTTTGATG ATGAATATTTTTTATTTTTATA TACATGATCGTATATAC SEQ ID NO: 453 GTATATACTTTTTATTTTTGATG ATAAATGTTTTTTATTTTTACA TATATGATCGTATATAC SEQ ID NO: 454 GGATATACTTTTTATTTTTGATG ATGAATATTTTTTATTTTTATA TACATGATCGTATATCC SEQ ID NO: 455 GGTGATACTTTTTATTTTTGATG ATGAATATTTTTTATTTTTATA TACATGATCGTATCACC SEQ ID NO: 456 GGTGATACTTTTTATTTTTGATG ATAAATGTTTTTTATTTTTAC ATATATGATCGTATCACC SEQ ID NO: 457 GGTGATCCTTTTTATTTTTGATG ATGAATATTTTTTATTTTTATA TACATGATCGGATCACC SEQ ID NO: 458 GATACTTTTTATTTTTGATATAA ATATATAATTTTTATTTTTATA TATATATATATCGTATC SEQ ID NO: 459 GATACTTTTTATTTTTGATAAAT GAATATATTTTTTATTTTTATA TATACATATATCGTATC SEQ ID NO: 460 GACACTTTTTATTTTTGATATAA ATATATAATTTTTATTTTTAT ATATATATATATCGTGTC SEQ ID NO: 461 GACACTTTTTATTTTTGATAAAT GAATATATTTTTTATTTTTAT ATATACATATATCGTGTC SEQ ID NO: 462 GACACTTTTTATTTTTGATATAA GTAAATATTTTTTATTTTTAT ATATACATATATCGTGTC SEQ ID NO: 463 GGATCTTTTTATTTTTGATATAA ATATATAATTTTTATTTTTATA TATATATATATCGATCC SEQ ID NO: 464 GGATCTTTTTATTTTTGATAAAT GAATATATTTTTTATTTTTATA TATACATATATCGATCC SEQ ID NO: 465 GGATCTTTTTATTTTTGATATAA GTAAATATTTTTTATTTTTATA TATACATATATCGATCC SEQ ID NO: 466 GCGTCTTTTTATTTTTGATATAA ATATATAATTTTTATTTTTATA TATATATATATCGACGC SEQ ID NO: 467 GCGTCTTTTTATTTTTGATAAAT GAATATATTTTTTATTTTTATA TATACATATATCGACGC SEQ ID NO: 468 GCGTCTTTTTATTTTTGATATAA GTAAATATTTTTTATTTTTATA TATACATATATCGACGC SEQ ID NO: 469 GTATATACATTTTTTATTTTTGA TATAAATATATAATTTTTATTT TTATATATATATATATCATGTAT ATAC SEQ ID NO: 470 GTATATACATTTTTTATTTTTGA TAAATGAATATATTTTTTATTT TTATATATACATATATCATGTAT ATAC SEQ ID NO: 471 GTATATACATTTTTTATTTTTGA TATAAGTAAATATTTTTTATTT TTATATATACATATATCATGTAT ATAC SEQ ID NO: 472 GGATATACATTTTTTATTTTTGA TATAAATATATAATTTTTATT TTTATATATATATATATCATGTA TATCC SEQ ID NO: 473 GGATATACACTTTTTATTTTTGA TATAAATATATAATTTTTATT TTTATATATATATATATCGTGTA TATCC SEQ ID NO: 474 GGATATACACTTTTTATTTTTGA TAAATGAATATATTTTTTATT TTTATATATACATATATCGTGTA TATCC SEQ ID NO: 475 GGATATACACTTTTTATTTTTGA TATAAGTAAATATTTTTTATT TTTATATATACATATATCGTGTA TATCC SEQ ID NO: 476 GGGTATATACTTTTTATTTTTGA TATAAATATATAATTTTTATT TTTATATATATATATATCGTATA TACCC SEQ ID NO: 477 GGGTATATACTTTTTATTTTTGA TAAATGAATATATTTTTTATT TTTATATATACATATATCGTATA TACCC SEQ ID NO: 478 GGGTATATACTTTTTATTTTTGA TATAAGTAAATATTTTTTATT TTTATATATACATATATCGTATA TACCC SEQ ID NO: 479 GTATACTTTTTATTTTTTATAAA TATATATTTTTTTATTTTTTAT ATATATATATAGTATAC SEQ ID NO: 480 GTGATCTTTTTATTTTTTATAAA TATATATTTTTTTATTTTTTAT ATATATATATAGATCAC SEQ ID NO: 481 GTATACTTTTTATTTTTGATATA AATATATTTTTTTATTTTTTAT ATATATATATCGTATAC SEQ ID NO: 482 GTATACTTTTTATTTTTGATATA TAAATATTTTTTTATTTTTTAT ATATATATATCGTATAC SEQ ID NO: 483 GTATACTTTTTATTTTTGATAAA TGAATATATTTTTATTTTTTAT ATACATATATCGTATAC SEQ ID NO: 484 GGATACTTTTTATTTTTTATAAA TATATATTTTTTTATTTTTTAT ATATATATATAGTATCC SEQ ID NO: 485 GTGATCTTTTTATTTTTGATATA AATATATTTTTTTATTTTTTAT ATATATATATCGATCAC SEQ ID NO: 486 GTGATCTTTTTATTTTTGATATA TAAATATTTTTTTATTTTTTAT ATATATATATCGATCAC SEQ ID NO: 487 GTGATCTTTTTATTTTTGATAAA TGAATATATTTTTATTTTTTAT ATACATATATCGATCAC SEQ ID NO: 488 GGATACTTTTTATTTTTGATATA AATATATTTTTTTATTTTTTAT ATATATATATCGTATCC SEQ ID NO: 489 GGATACTTTTTATTTTTGATATA TAAATATTTTTTTATTTTTTAT ATATATATATCGTATCC SEQ ID NO: 490 GGATACTTTTTATTTTTGATAAA TGAATATATTTTTATTTTTTAT ATACATATATCGTATCC SEQ ID NO: 491 GCGATCTTTTTATTTTTTATAAA TATATATTTTTTTATTTTTTAT ATATATATATAGATCGC SEQ ID NO: 492 GCGATCTTTTTATTTTTGATATA AATATATTTTTTTATTTTTTAT ATATATATATCGATCGC SEQ ID NO: 493 GCGATCTTTTTATTTTTGATATA TAAATATTTTTTTATTTTTTAT ATATATATATCGATCGC SEQ ID NO: 494 GCGATCTTTTTATTTTTGATAAA TGAATATATTTTTATTTTTTAT ATACATATATCGATCGC SEQ ID NO: 495 GATATATCACTTTTTATTTTTTA TAAATATATATTTTTTTATTTT TTATATATATATATAGTGATATA TC SEQ ID NO: 496 GTATATACATTTTTTATTTTTGA TATAAATATATTTTTTTATTTT TTATATATATATATCATGTATAT AC SEQ ID NO: 497 GTATATACATTTTTTATTTTTGA TATATAAATATTTTTTTATTTT TTATATATATATATCATGTATAT AC SEQ ID NO: 498 GGATATACACTTTTTATTTTTTA TAAATATATATTTTTTTATTTT TTATATATATATATAGTGTATAT CC SEQ ID NO: 499 GGATATACATTTTTTATTTTTGA TATAAATATATTTTTTTATTTT TTATATATATATATCATGTATAT CC SEQ ID NO: 500 GGATATACATTTTTTATTTTTGA TATATAAATATTTTTTTATTTT TTATATATATATATCATGTATAT CC SEQ ID NO: 501 GGATATACATTTTTTATTTTTGA TAAATGAATATATTTTTATTT TTTATATACATATATCATGTATA TCC SEQ ID NO: 502 GGGTATATACTTTTTATTTTTTA TAAATATATATTTTTTTATTTT TTATATATATATATAGTATATAC CC SEQ ID NO: 503 GGATATACACTTTTTATTTTTGA TATAAATATATTTTTTTATTTT TTATATATATATATCGTGTATAT CC SEQ ID NO: 504 GGATATACACTTTTTATTTTTGA TATATAAATATTTTTTTATTTT TTATATATATATATCGTGTATAT CC SEQ ID NO: 505 GGATATACACTTTTTATTTTTGA TAAATGAATATATTTTTATTT TTTATATACATATATCGTGTATA TCC SEQ ID NO: 506 GGGTATATACTTTTTATTTTTGA TATAAATATATTTTTTTATTTT TTATATATATATATCGTATATAC CC SEQ ID NO: 507 GGGTATATACTTTTTATTTTTGA TATATAAATATTTTTTTATTTT TTATATATATATATCGTATATAC CC SEQ ID NO: 508 GGGTATATACTTTTTATTTTTGA TAAATGAATATATTTTTATTT TTTATATACATATATCGTATATA CCC SEQ ID NO: 509 GATATACTTTTTATTTTTGATAA ATATATAATTTTTATTTTTATA TATATATATCGTATATC SEQ ID NO: 510 GTGATACTTTTTATTTTTGATAA ATATATAATTTTTATTTTTATA TATATATATCGTATCAC SEQ ID NO: 511 GGTATACTTTTTATTTTTGATAA ATATATAATTTTTATTTTTATA TATATATATCGTATACC SEQ ID NO: 512 GGTGTACTTTTTATTTTTGATAA ATATATAATTTTTATTTTTATA TATATATATCGTACACC SEQ ID NO: 513 GTATATACATTTTTTATTTTTGA TAAATATATAATTTTTATTTTT ATATATATATATCATGTATATAC SEQ ID NO: 514 GGATATACATTTTTTATTTTTGA TAAATATATAATTTTTATTTTT ATATATATATATCATGTATATCC SEQ ID NO: 515 GGATATACACTTTTTATTTTTGA TAAATATATAATTTTTATTTT TATATATATATATCGTGTATATC C SEQ ID NO: 516 GGGTATATACTTTTTATTTTTGA TAAATATATAATTTTTATTTTT ATATATATATATCGTATATACCC SEQ ID NO: 517 GTATATACTTTTTATTTTTGATA AATATATTTTTTTATTTTTTAT ATATATATCGTATATAC SEQ ID NO: 518 GTATATACTTTTTATTTTTGATA AATGTATTTTTTTATTTTTTAT ACATATATCGTATATAC SEQ ID NO: 519 GGATATACTTTTTATTTTTGATA AATATATTTTTTTATTTTTTAT ATATATATCGTATATCC SEQ ID NO: 520 GGATATACTTTTTATTTTTGATA AATGTATTTTTTTATTTTTTAT ACATATATCGTATATCC SEQ ID NO: 521 GGATATACTTTTTATTTTTGATG ATAAATGTTTTTTATTTTTAC ATATATGATCGTATATCC SEQ ID NO: 522 GGTGATACTTTTTATTTTTGATA AATATATTTTTTTATTTTTTAT ATATATATCGTATCACC SEQ ID NO: 523 GGTGATACTTTTTATTTTTGATA AATGTATTTTTTTATTTTTTAT ACATATATCGTATCACC SEQ ID NO: 524 GGTGATCCTTTTTATTTTTGATA AATATATTTTTTTATTTTTTAT ATATATATCGGATCACC SEQ ID NO: 525 GGTGATCCTTTTTATTTTTGATA AATGTATTTTTTTATTTTTTAT ACATATATCGGATCACC SEQ ID NO: 526 GGTGATCCTTTTTATTTTTGATG ATAAATGTTTTTTATTTTTACA TATATGATCGGATCACC SEQ ID NO: 527 GTATATACATTTTTTATTTTTGA TAAATATATTTTTTTATTTTTT ATATATATATCATGTATATAC SEQ ID NO: 528 GTATATACATTTTTTATTTTTGA TAAATGTATTTTTTTATTTTTT ATACATATATCATGTATATAC SEQ ID NO: 529 GTATATACATTTTTTATTTTTGA TGATAAATGTTTTTTATTTTTA CATATATGATCATGTATATAC SEQ ID NO: 530 GGATATACATTTTTTATTTTTGA TAAATATATTTTTTTATTTTTT ATATATATATCATGTATATCC SEQ ID NO: 531 GGATATACACTTTTTATTTTTGA TAAATATATTTTTTTATTTTTT ATATATATATCGTGTATATCC SEQ ID NO: 532 GGATATACACTTTTTATTTTTGA TAAATGTATTTTTTTATTTTTT ATACATATATCGTGTATATCC SEQ ID NO: 533 GGATATACACTTTTTATTTTTGA TGATAAATGTTTTTTATTTTT ACATATATGATCGTGTATATCC SEQ ID NO: 534 GGGTATATACTTTTTATTTTTGA TAAATATATTTTTTTATTTTTT ATATATATATCGTATATACCC SEQ ID NO: 535 GGGTATATACTTTTTATTTTTGA TAAATGTATTTTTTTATTTTTT ATACATATATCGTATATACCC SEQ ID NO: 536 GGGTATATACTTTTTATTTTTGA TGATAAATGTTTTTTATTTTTA CATATATGATCGTATATACCC SEQ ID NO: 537 GTATATACATTTTTTATTTTTGA TAAATATTTTTTTATTTTTTAT ATATATCATGTATATAC SEQ ID NO: 538 GTATATACATTTTTTATTTTTGA TAAATGTTTTTTTATTTTTTAC ATATATCATGTATATAC SEQ ID NO: 539 GGATATACATTTTTTATTTTTGA TAAATATTTTTTTATTTTTTAT ATATATCATGTATATCC SEQ ID NO: 540 GGATATACATTTTTTATTTTTGA TAAATGTTTTTTTATTTTTTAC ATATATCATGTATATCC SEQ ID NO: 541 GGATATACATTTTTTATTTTTGA TGATGAATTTTTTATTTTTATA CATGATCATGTATATCC SEQ ID NO: 542 GGATATACACTTTTTATTTTTGA TAAATATTTTTTTATTTTTTAT ATATATCGTGTATATCC SEQ ID NO: 543 GGATATACACTTTTTATTTTTGA TAAATGTTTTTTTATTTTTTAC ATATATCGTGTATATCC SEQ ID NO: 544 GGGTATATACTTTTTATTTTTGA TAAATATTTTTTTATTTTTTAT ATATATCGTATATACCC SEQ ID NO: 545 GGGTATATACTTTTTATTTTTGA TAAATGTTTTTTTATTTTTTAC ATATATCGTATATACCC SEQ ID NO: 546 GGATGTACACTTTTTATTTTTGA TAAATATTTTTTTATTTTTTAT ATATATCGTGTACATCC SEQ ID NO: 547 GGATGTACACTTTTTATTTTTGA TAAATGTTTTTTTATTTTTTAC ATATATCGTGTACATCC SEQ ID NO: 548 GTACATATATTTTTTTATTTTTG ATAAATATTTTTTTATTTTTTA TATATATCAATATATGTAC SEQ ID NO: 549 GTACATATATTTTTTTATTTTTG ATAAATGTTTTTTTATTTTTTA CATATATCAATATATGTAC SEQ ID NO: 550 GGTACATATATTTTTTATTTTTG ATAAATATTTTTTTATTTTTTA TATATATCATATATGTACC SEQ ID NO: 551 GGTACATATATTTTTTATTTTTG ATAAATGTTTTTTTATTTTTTA CATATATCATATATGTACC SEQ ID NO: 552 CGATCATATATTTTTTTATTTTT GATAAATATTTTTTTATTTTTT ATATATATCAATATATGATCG SEQ ID NO: 553 CGATCATATATTTTTTTATTTTT GATAAATGTTTTTTTATTTTTT ACATATATCAATATATGATCG SEQ ID NO: 554 CGATCATATATTTTTTTATTTTT GATGATGAATTTTTTATTTTTA TACATGATCAATATATGATCG SEQ ID NO: 555 CGATCATATATTTTTTTATTTTT GATGATAAATTTTTTATTTTTA TATATGATCAATATATGATCG SEQ ID NO: 556 GTATATACTTTTTATTTTTGATA TAAATATATAATTTTTATTTTT ATATATATATATATCGTATATAC SEQ ID NO: 557 GTATATACTTTTTATTTTTGATA AATGAATATATTTTTTATTTTT ATATATACATATATCGTATATAC SEQ ID NO: 558 GGATATACTTTTTATTTTTGATA TAAATATATAATTTTTATTTTT ATATATATATATATCGTATATCC SEQ ID NO: 559 GGATATACTTTTTATTTTTGATA AATGAATATATTTTTTATTTTT ATATATACATATATCGTATATCC SEQ ID NO: 560 GGTGATACTTTTTATTTTTGATA TAAATATATAATTTTTATTTTT ATATATATATATATCGTATCACC SEQ ID NO: 561 GGTGATACTTTTTATTTTTGATA AATGAATATATTTTTTATTTTT ATATATACATATATCGTATCACC SEQ ID NO: 562 GGTGATCCTTTTTATTTTTGATA TAAATATATAATTTTTATTTTT ATATATATATATATCGGATCACC SEQ ID NO: 563 GGTGATCCTTTTTATTTTTGATA AATGAATATATTTTTTATTTTT ATATATACATATATCGGATCACC SEQ ID NO: 564 GTATATACTTTTTATTTTTGATA TAAGTAAATATTTTTTATTTTT ATATATACATATATCGTATATAC SEQ ID NO: 565 GGATATACTTTTTATTTTTGATA TAAGTAAATATTTTTTATTTTT ATATATACATATATCGTATATCC SEQ ID NO: 566 GGTGATACTTTTTATTTTTGATA TAAGTAAATATTTTTTATTTTT ATATATACATATATCGTATCACC SEQ ID NO: 567 GGTGATCCTTTTTATTTTTGATA TAAGTAAATATTTTTTATTTTT ATATATACATATATCGGATCACC SEQ ID NO: 568 GTATATACTTTTTATTTTTTATA AATATATATTTTTTTATTTTTT ATATATATATATAGTATATAC SEQ ID NO: 569 GTATATACTTTTTATTTTTGATA TAAATATATTTTTTTATTTTTT ATATATATATATCGTATATAC SEQ ID NO: 570 GTATATACTTTTTATTTTTGATA AATGAATATATTTTTATTTTTT ATATACATATATCGTATATAC SEQ ID NO: 571 GGATATACTTTTTATTTTTTATA AATATATATTTTTTTATTTTTT ATATATATATATAGTATATCC SEQ ID NO: 572 GGATATACTTTTTATTTTTGATA TAAATATATTTTTTTATTTTTT ATATATATATATCGTATATCC SEQ ID NO: 573 GGATATACTTTTTATTTTTGATA AATGAATATATTTTTATTTTTT ATATACATATATCGTATATCC SEQ ID NO: 574 GGTGATACTTTTTATTTTTTATA AATATATATTTTTTTATTTTTT ATATATATATATAGTATCACC SEQ ID NO: 575 GGTGATACTTTTTATTTTTGATA TAAATATATTTTTTTATTTTTT ATATATATATATCGTATCACC SEQ ID NO: 576 GGTGATACTTTTTATTTTTGATA AATGAATATATTTTTATTTTTT ATATACATATATCGTATCACC SEQ ID NO: 577 GGTGATCCTTTTTATTTTTTATA AATATATATTTTTTTATTTTTT ATATATATATATAGGATCACC SEQ ID NO: 578 GGTGATCCTTTTTATTTTTGATA TAAATATATTTTTTTATTTTTT ATATATATATATCGGATCACC SEQ ID NO: 579 GGTGATCCTTTTTATTTTTGATA AATGAATATATTTTTATTTTTT ATATACATATATCGGATCACC SEQ ID NO: 580 GTATATACTTTTTATTTTTGATA TATAAATATTTTTTTATTTTTT ATATATATATATCGTATATAC SEQ ID NO: 581 GGATATACTTTTTATTTTTGATA TATAAATATTTTTTTATTTTTT ATATATATATATCGTATATCC SEQ ID NO: 582 GGTGATACTTTTTATTTTTGATA TATAAATATTTTTTTATTTTTT ATATATATATATCGTATCACC SEQ ID NO: 583 GGTGATCCTTTTTATTTTTGATA TATAAATATTTTTTTATTTTTT ATATATATATATCGGATCACC SEQ ID NO: 584 GATACAAAAAAAAAAATATATAT ATATATATAAAAAAAAAAA ATATATATATATATAGTATC SEQ ID NO: 585 GACACAAAAAAAAAAAGATATAT ATATATATAAAAAAAAAAA ATATATATATATATCGTGTC SEQ ID NO: 586 GATATACAAAAAAAAAAATATAT ATATATATAAAAAAAAAAA ATATATATATATAGTATATC SEQ ID NO: 587 GATATATAAAAAAAAAAAGATAT ATGTATATAAAAAAAAAAA ATATACATATATCATATATC SEQ ID NO: 588 GATATACAAAAAAAAAAAGATAT ATATATATAAAAAAAAAAA ATATATATATATCGTATATC SEQ ID NO: 589 GGTATACAAAAAAAAAAATATAT ATATATATAAAAAAAAAAA ATATATATATATAGTATACC SEQ ID NO: 590 GATATATCACAAAAAAAAAAATA TATATATAAAAAAAAAAAA TATATATATAGTGATATATC SEQ ID NO: 591 GTATATACATAAAAAAAAAAAGA TATATGTAAAAAAAAAAAA TACATATATCATGTATATAC SEQ ID NO: 592 GGATATACATAAAAAAAAAAAGA TATATGTAAAAAAAAAAA ATACATATATCATGTATATCC SEQ ID NO: 593 GGATATACATAAAAAAAAAAAGA TCATGTATAAAAAAAAAAA ATACATGATCATGTATATCC SEQ ID NO: 594 GGGTATATACAAAAAAAAAAATA TATATATAAAAAAAAAAAA TATATATATAGTATATACCC SEQ ID NO: 595 GTATATACAAAAAAAAAAATATA TATATATATATAAAAAAAA AAAATATATATATATATAGTATA TAC SEQ ID NO: 596 GTATATACAAAAAAAAAAAGATA TATATATATATAAAAAAAA AAAATATATATATATATCGTATA TAC SEQ ID NO: 597 GGATATACAAAAAAAAAAATATA TATATATATATAAAAAAAA AAAATATATATATATATAGTATA TCC SEQ ID NO: 598 GGATATACAAAAAAAAAAAGATA TATATATATATAAAAAAAA AAAATATATATATATATCGTATA TCC SEQ ID NO: 599 GTATATACAAAAAAAAAAATATA TATATATATAAAAAAAAAA AATATATATATATATAGTATATA C SEQ ID NO: 600 GTATATACAAAAAAAAAAAGATA TATATATATAAAAAAAAAA AATATATATATATATCGTATATA C SEQ ID NO: 601 GGATATACAAAAAAAAAAATATA TATATATATAAAAAAAAAA AATATATATATATATAGTATATC C SEQ ID NO: 602 GGATATACAAAAAAAAAAAGATA TATATATATAAAAAAAAAA AATATATATATATATCGTATATC C SEQ ID NO: 603 GATATATCACAAAAAAAAAAATA TATATATATAAAAAAAAAA AATATATATATATAGTGATATAT C SEQ ID NO: 604 GGATATACATAAAAAAAAAAAGA TATATATATAAAAAAAAAA AATATATATATATCATGTATATC C SEQ ID NO: 605 GTACATATATTAAAAAAAAAAAG ATATATATAAAAAAAAAAA ATATATATATCAATATATGTAC SEQ ID NO: 606 GATGTATATACAAAAAAAAAAAT ATATATATAAAAAAAAAAA ATATATATATAGTATATACATC SEQ ID NO: 607 CGATCATATATTAAAAAAAAAAA GATATATATAAAAAAAAAA AATATATATATCAATATATGATC G SEQ ID NO: 608 CGATCATATATTAAAAAAAAAAA GATATATGTAAAAAAAAAA AATACATATATCAATATATGATC G SEQ ID NO: 609 GATACAAAAAAAAAAATATAAAT ATATATATAAAAAAAAAAA ATATATATATATATAGTATC SEQ ID NO: 610 GGATCAAAAAAAAAAATATAAAT ATATATATAAAAAAAAAAA ATATATATATATATAGATCC SEQ ID NO: 611 GACACAAAAAAAAAAAGATAAAT ATATATATAAAAAAAAAA AATATATATATATATCGTGTC SEQ ID NO: 612 GACACAAAAAAAAAAAGATGATG TATATATAAAAAAAAAAA ATATATATACATGATCGTGTC SEQ ID NO: 613 GCGTCAAAAAAAAAAAGATAAAT ATATATATAAAAAAAAAAA ATATATATATATATCGACGC SEQ ID NO: 614 GATATACAAAAAAAAAAATATAA ATATATATAAAAAAAAAAA ATATATATATATAGTATATC SEQ ID NO: 615 GTATATACATAAAAAAAAAAAGA TAAATGTAAAAAAAAAAA ATACATATATCATGTATATAC SEQ ID NO: 616 GTATATACATAAAAAAAAAAAGA TGATATATAAAAAAAAAAA ATATATGATCATGTATATAC SEQ ID NO: 617 GGATATACATAAAAAAAAAAAGA TAAATATAAAAAAAAAAA ATATATATATCATGTATATCC SEQ ID NO: 618 GGATATACATAAAAAAAAAAAGA TGATATATAAAAAAAAAA AATATATGATCATGTATATCC SEQ ID NO: 619 GTATATACAAAAAAAAAAATATA AATATATATATAAAAAAAA AAAATATATATATATATAGTATA TAC SEQ ID NO: 620 GTATATACAAAAAAAAAAAGATA AATATATATATAAAAAAAA AAAATATATATATATATCGTATA TAC SEQ ID NO: 621 GGATATACAAAAAAAAAAATATA AATATATATATAAAAAAAA AAAATATATATATATATAGTATA TCC SEQ ID NO: 622 GGATATACAAAAAAAAAAAGATA AATATATATATAAAAAAAA AAAATATATATATATATCGTATA TCC SEQ ID NO: 623 GTATATACAAAAAAAAAAAGATA AATGTATATAAAAAAAAAA AATATATACATATATCGTATATA C SEQ ID NO: 624 GGATATACAAAAAAAAAAAGATA AATGTATATAAAAAAAAA AAATATATACATATATCGTATAT CC SEQ ID NO: 625 GGTGATACAAAAAAAAAAAGATG ATGTATATATAAAAAAAAA AAATATATACATGATCGTATCAC C SEQ ID NO: 626 GATATATCACAAAAAAAAAAATA TAAATATATATAAAAAAAA AAAATATATATATATAGTGATAT ATC SEQ ID NO: 627 GTATATACATAAAAAAAAAAAGA TAAATATATAAAAAAAAAA AATATATATATATCATGTATATA C SEQ ID NO: 628 GTATATACATAAAAAAAAAAAGA TGATATATGTAAAAAAAAA AAACATATATGATCATGTATATA C SEQ ID NO: 629 GGATATACATAAAAAAAAAAAGA TAAATATATAAAAAAAAA AAATATATATATATCATGTATAT CC SEQ ID NO: 630 GTACATATATTAAAAAAAAAAAG ATAAATATAAAAAAAAAAA ATATATATATCAATATATGTAC SEQ ID NO: 631 GTACATATATTAAAAAAAAAAAG ATAAATGTAAAAAAAAAAA ATACATATATCAATATATGTAC SEQ ID NO: 632 GTACATATATTAAAAAAAAAAAG ATGATATATAAAAAAAAAA AATATATGATCAATATATGTAC SEQ ID NO: 633 GGATATACATAAAAAAAAAAAGA TGATGAATAAAAAAAAAA AATACATGATCATGTATATCC SEQ ID NO: 634 GTATATACATAAAAAAAAAAAGA TAAATGTTAAAAAAAAAAA TACATATATCATGTATATAC SEQ ID NO: 635 GATACAAAAAAAAAAAGATATAA ATATATAAAAAAAAAAAA AATATATATATATATCGTATC SEQ ID NO: 636 GATACAAAAAAAAAAAGATGATA TAAGTACTAAAAAAAAAA AAGTACATATATGATCGTATC SEQ ID NO: 637 GACACAAAAAAAAAAAGATAAAT GAATATATAAAAAAAAAA AATATATACATATATCGTGTC SEQ ID NO: 638 GGATATACAAAAAAAAAAAGATA TAAGTAAATATAAAAAAA AAAAATATATACATATATCGTAT ATCC SEQ ID NO: 639 GGATATACAAAAAAAAAAAGATG ATATAAGTACTAAAAAAAA AAAAGTACATATATGATCGTATA TCC SEQ ID NO: 640 GTATATACAAAAAAAAAAAGATA TAAGTAAATATAAAAAAAA AAAATATATACATATATCGTATA TAC SEQ ID NO: 641 GTATATACAAAAAAAAAAAGATG ATATAAGTACTAAAAAAAA AAAAGTACATATATGATCGTATA TAC SEQ ID NO: 642 GGATATACAAAAAAAAAAAGATA AATGAATATAAAAAAAAA AAATATATACATATATCGTATAT CC SEQ ID NO: 643 GTATATACAAAAAAAAAAAGATA AATGAATATAAAAAAAAA AAATATATACATATATCGTATAT AC SEQ ID NO: 644 GTATATACAAAAAAAAAAAGATG ATATAAGTACAAAAAAAAA AAGTACATATATGATCGTATATA C SEQ ID NO: 645 GTATATACAAAAAAAAAAATATA AATATATATTAAAAAAAAA AATATATATATATATAGTATATA C SEQ ID NO: 646 GTATATACATAAAAAAAAAAAGA TGATGTAAATATAAAAAAA AAAAATATATACATGATCATGTA TATAC SEQ ID NO: 647 GATATACAAAAAAAAAAAGATAA ATATATAAAAAAAAAAAA AATATATATATATCGTATATC SEQ ID NO: 648 GTGATACAAAAAAAAAAAGATAA ATATATAAAAAAAAAAAA AATATATATATATCGTATCAC SEQ ID NO: 649 GGTATACAAAAAAAAAAAGATAA ATATATAAAAAAAAAAAA AATATATATATATCGTATACC SEQ ID NO: 650 GGATATACATAAAAAAAAAAAGA TAAATGAATAAAAAAAAA AAATATACATATATCATGTATAT CC SEQ ID NO: 651 GTATATACATAAAAAAAAAAAGA TAAATGTATTAAAAAAAAA AATATACATATATCATGTATATA C SEQ ID NO: 652 GTATATACATAAAAAAAAAAAGA TGATAAATGTAAAAAAAAA AAACATATATGATCATGTATATA C SEQ ID NO: 653 GATACTTTTTATTTTTTATATAT ATATATATTTTTTATTTTTATA T*A*T*A*T*A*T*A*T*A*T*A *G*T*A*T*C SEQ ID NO: 654 GACACTTTTTATTTTTTATATAT ATATATATTTTTTATTTTTATA T*A*T*A*T*A*T*A*T*A*T*A *G*T*G*T*C SEQ ID NO: 655 GATACTTTTTATTTTTGATATAT ATATATATTTTTTATTTTTATA T*A*T*A*T*A*T*A*T*A*T*C *G*T*A*T*C SEQ ID NO: 656 GGATCTTTTTATTTTTTATATAT ATATATATTTTTTATTTTTATA T*A*T*A*T*A*T*A*T*A*T*A *G*A*T*C*C SEQ ID NO: 657 GACACTTTTTATTTTTGATATAT ATATATATTTTTTATTTTTATA T*A*T*A*T*A*T*A*T*A*T*C *G*T*G*T*C SEQ ID NO: 658 GGATCTTTTTATTTTTGATATAT ATATATATTTTTTATTTTTATA T*A*T*A*T*A*T*A*T*A*T*C *G*A*T*C*C SEQ ID NO: 659 GCGTCTTTTTATTTTTTATATAT ATATATATTTTTTATTTTTATA T*A*T*A*T*A*T*A*T*A*T*A *G*A*C*G*C SEQ ID NO: 660 GCGTCTTTTTATTTTTGATATAT ATATATATTTTTTATTTTTATA T*A*T*A*T*A*T*A*T*A*T*C *G*A*C*G*C SEQ ID NO: 661 GTATACTTTTTATTTTTTATATA TATATATATTTTTATTTTTTAT A*T*A*T*A*T*A*T*A*T*A*G *T*A*T*A*C SEQ ID NO: 662 GTGATCTTTTTATTTTTTATATA TATATATATTTTTATTTTTTAT A*T*A*T*A*T*A*T*A*T*A*G *A*T*C*A*C SEQ ID NO: 663 GTATACTTTTTATTTTTGATATA TATATATATTTTTATTTTTTAT A*T*A*T*A*T*A*T*A*T*C*G *T*A*T*A*C SEQ ID NO: 664 GTATACTTTTTATTTTTGATATA TGTATATATTTTTATTTTTTAT A*T*A*C*A*T*A*T*A*T*C*G *T*A*T*A*C SEQ ID NO: 665 GGATACTTTTTATTTTTTATATA TATATATATTTTTATTTTTTAT A*T*A*T*A*T*A*T*A*T*A*G *T*A*T*C*C SEQ ID NO: 666 GTGATCTTTTTATTTTTGATATA TATATATATTTTTATTTTTTAT A*T*A*T*A*T*A*T*A*T*C*G *A*T*C*A*C SEQ ID NO: 667 GTGATCTTTTTATTTTTGATATA TGTATATATTTTTATTTTTTAT A*T*A*C*A*T*A*T*A*T*C*G *A*T*C*A*C SEQ ID NO: 668 GGATACTTTTTATTTTTGATATA TATATATATTTTTATTTTTTAT A*T*A*T*A*T*A*T*A*T*C*G *T*A*T*C*C SEQ ID NO: 669 GGATACTTTTTATTTTTGATATA TGTATATATTTTTATTTTTTAT A*T*A*C*A*T*A*T*A*T*C*G *T*A*T*C*C SEQ ID NO: 670 GCGATCTTTTTATTTTTTATATA TATATATATTTTTATTTTTTAT A*T*A*T*A*T*A*T*A*T*A*G *A*T*C*G*C SEQ ID NO: 671 GCGATCTTTTTATTTTTGATATA TATATATATTTTTATTTTTTAT A*T*A*T*A*T*A*T*A*T*C*G *A*T*C*G*C SEQ ID NO: 672 GCGATCTTTTTATTTTTGATATA TGTATATATTTTTATTTTTTAT A*T*A*C*A*T*A*T*A*T*C*G *A*T*C*G*C SEQ ID NO: 673 GATATACTTTTTATTTTTTATAT ATATATATTTTTTATTTTTATA T*A*T*A*T*A*T*A*T*A*G*T *A*T*A*T*C SEQ ID NO: 674 GATATATTTTTTATTTTTGATAT ATATATATTTTTTATTTTTATA T*A*T*A*T*A*T*A*T*C*A*T *A*T*A*T*C SEQ ID NO: 675 GATATATTTTTTATTTTTGATAT ATGTATATTTTTTATTTTTATA T*A*C*A*T*A*T*A*T*C*A*T *A*T*A*T*C SEQ ID NO: 676 GTGATACTTTTTATTTTTTATAT ATATATATTTTTTATTTTTATA T*A*T*A*T*A*T*A*T*A*G*T *A*T*C*A*C SEQ ID NO: 677 GATATACTTTTTATTTTTGATAT ATATATATTTTTTATTTTTATA T*A*T*A*T*A*T*A*T*C*G*T *A*T*A*T*C SEQ ID NO: 678 GATATACTTTTTATTTTTGATAT ATGTATATTTTTTATTTTTATA T*A*C*A*T*A*T*A*T*C*G*T *A*T*A*T*C SEQ ID NO: 679 GGTATACTTTTTATTTTTTATAT ATATATATTTTTTATTTTTATA T*A*T*A*T*A*T*A*T*A*G*T *A*T*A*C*C SEQ ID NO: 680 GTGATACTTTTTATTTTTGATAT ATATATATTTTTTATTTTTATA T*A*T*A*T*A*T*A*T*C*G*T *A*T*C*A*C SEQ ID NO: 681 GTGATACTTTTTATTTTTGATAT ATGTATATTTTTTATTTTTATA T*A*C*A*T*A*T*A*T*C*G*T *A*T*C*A*C SEQ ID NO: 682 GGTATACTTTTTATTTTTGATAT ATATATATTTTTTATTTTTATA T*A*T*A*T*A*T*A*T*C*G*T *A*T*A*C*C SEQ ID NO: 683 GGTATACTTTTTATTTTTGATAT ATGTATATTTTTTATTTTTATA T*A*C*A*T*A*T*A*T*C*G*T *A*T*A*C*C SEQ ID NO: 684 GGTGTACTTTTTATTTTTTATAT ATATATATTTTTTATTTTTATA T*A*T*A*T*A*T*A*T*A*G*T *A*C*A*C*C SEQ ID NO: 685 GGTGTACTTTTTATTTTTGATAT ATATATATTTTTTATTTTTATA T*A*T*A*T*A*T*A*T*C*G*T *A*C*A*C*C SEQ ID NO: 686 GGTGTACTTTTTATTTTTGATAT ATGTATATTTTTTATTTTTATA T*A*C*A*T*A*T*A*T*C*G*T *A*C*A*C*C SEQ ID NO: 687 GTATATACTTTTTATTTTTTATA TATATATATTTTTATTTTTTAT A*T*A*T*A*T*A*T*A*G*T*A *T*A*T*A*C SEQ ID NO: 688 GTATATACTTTTTATTTTTGATA TATATATATTTTTATTTTTTAT A*T*A*T*A*T*A*T*C*G*T*A *T*A*T*A*C SEQ ID NO: 689 GTATATACTTTTTATTTTTGATA TATGTATATTTTTATTTTTTAT A*C*A*T*A*T*A*T*C*G*T*A *T*A*T*A*C SEQ ID NO: 690 GTATATACTTTTTATTTTTGATC ATGTATATTTTTTATTTTTATA T*A*C*A*T*G*A*T*C*G*T*A *T*A*T*A*C SEQ ID NO: 691 GTATATACTTTTTATTTTTGATC ATATATGTTTTTTATTTTTACA T*A*T*A*T*G*A*T*C*G*T*A *T*A*T*A*C SEQ ID NO: 692 GGATATACTTTTTATTTTTTATA TATATATATTTTTATTTTTTAT A*T*A*T*A*T*A*T*A*G*T*A *T*A*T*C*C SEQ ID NO: 693 GGATATACTTTTTATTTTTGATA TATATATATTTTTATTTTTTAT A*T*A*T*A*T*A*T*C*G*T*A *T*A*T*C*C SEQ ID NO: 694 GGATATACTTTTTATTTTTGATA TATGTATATTTTTATTTTTTAT A*C*A*T*A*T*A*T*C*G*T*A *T*A*T*C*C SEQ ID NO: 695 GGATATACTTTTTATTTTTGATC ATGTATATTTTTTATTTTTATA T*A*C*A*T*G*A*T*C*G*T*A *T*A*T*C*C SEQ ID NO: 696 GGATATACTTTTTATTTTTGATC ATATATGTTTTTTATTTTTACA T*A*T*A*T*G*A*T*C*G*T*A *T*A*T*C*C SEQ ID NO: 697 GGTGATACTTTTTATTTTTTATA TATATATATTTTTATTTTTTAT A*T*A*T*A*T*A*T*A*G*T*A *T*C*A*C*C SEQ ID NO: 698 GGTGATACTTTTTATTTTTGATA TATATATATTTTTATTTTTTAT A*T*A*T*A*T*A*T*C*G*T*A *T*C*A*C*C SEQ ID NO: 699 GGTGATACTTTTTATTTTTGATA TATGTATATTTTTATTTTTTAT A*C*A*T*A*T*A*T*C*G*T*A *T*C*A*C*C SEQ ID NO: 700 GGTGATACTTTTTATTTTTGATC ATGTATATTTTTTATTTTTATA T*A*C*A*T*G*A*T*C*G*T*A *T*C*A*C*C SEQ ID NO: 701 GGTGATACTTTTTATTTTTGATC ATATATGTTTTTTATTTTTACA T*A*T*A*T*G*A*T*C*G*T*A *T*C*A*C*C SEQ ID NO: 702 GGTGATCCTTTTTATTTTTTATA TATATATATTTTTATTTTTTAT A*T*A*T*A*T*A*T*A*G*G*A *T*C*A*C*C SEQ ID NO: 703 GGTGATCCTTTTTATTTTTGATA TATATATATTTTTATTTTTTAT A*T*A*T*A*T*A*T*C*G*G*A *T*C*A*C*C SEQ ID NO: 704 GGTGATCCTTTTTATTTTTGATA TATGTATATTTTTATTTTTTAT A*C*A*T*A*T*A*T*C*G*G*A *T*C*A*C*C SEQ ID NO: 705 GGTGATCCTTTTTATTTTTGATC ATGTATATTTTTTATTTTTATA T*A*C*A*T*G*A*T*C*G*G*A *T*C*A*C*C SEQ ID NO: 706 GGTGATCCTTTTTATTTTTGATC ATATATGTTTTTTATTTTTACA T*A*T*A*T*G*A*T*C*G*G*A *T*C*A*C*C SEQ ID NO: 707 GATATATCACTTTTTATTTTTTA TATATATATTTTTATTTTTTAT A*T*A*T*A*T*A*G*T*G*A*T *A*T*A*T*C SEQ ID NO: 708 GTATATACATTTTTTATTTTTGA TATATATATTTTTATTTTTTAT A*T*A*T*A*T*C*A*T*G*T*A *T*A*T*A*C SEQ ID NO: 709 GTATATACATTTTTTATTTTTGA TATATGTATTTTTATTTTTTAC A*T*A*T*A*T*C*A*T*G*T*A *T*A*T*A*C SEQ ID NO: 710 GTATATACATTTTTTATTTTTGA TCATGTATTTTTTATTTTTATA C*A*T*G*A*T*C*A*T*G*T*A *T*A*T*A*C SEQ ID NO: 711 GTATATACATTTTTTATTTTTGA TCATATATTTTTTATTTTTATA T*A*T*G*A*T*C*A*T*G*T*A *T*A*T*A*C SEQ ID NO: 712 GGATATACACTTTTTATTTTTTA TATATATATTTTTATTTTTTAT A*T*A*T*A*T*A*G*T*G*T*A *T*A*T*C*C SEQ ID NO: 713 GGATATACATTTTTTATTTTTGA TATATATATTTTTATTTTTTAT A*T*A*T*A*T*C*A*T*G*T*A *T*A*T*C*C SEQ ID NO: 714 GGATATACATTTTTTATTTTTGA TATATGTATTTTTATTTTTTAC A*T*A*T*A*T*C*A*T*G*T*A *T*A*T*C*C SEQ ID NO: 715 GGATATACATTTTTTATTTTTGA TCATGTATTTTTTATTTTTATA C*A*T*G*A*T*C*A*T*G*T*A *T*A*T*C*C SEQ ID NO: 716 GGATATACATTTTTTATTTTTGA TCATATATTTTTTATTTTTATA T*A*T*G*A*T*C*A*T*G*T*A *T*A*T*C*C SEQ ID NO: 717 GGGTATATACTTTTTATTTTTTA TATATATATTTTTATTTTTTAT A*T*A*T*A*T*A*G*T*A*T*A *T*A*C*C*C SEQ ID NO: 718 GGATATACACTTTTTATTTTTGA TATATATATTTTTATTTTTTAT A*T*A*T*A*T*C*G*T*G*T*A *T*A*T*C*C SEQ ID NO: 719 GGATATACACTTTTTATTTTTGA TATATGTATTTTTATTTTTTAC A*T*A*T*A*T*C*G*T*G*T*A *T*A*T*C*C SEQ ID NO: 720 GGATATACACTTTTTATTTTTGA TCATGTATTTTTTATTTTTATA C*A*T*G*A*T*C*G*T*G*T*A *T*A*T*C*C SEQ ID NO: 721 GGATATACACTTTTTATTTTTGA TCATATATTTTTTATTTTTATA T*A*T*G*A*T*C*G*T*G*T*A *T*A*T*C*C SEQ ID NO: 722 GGGTATATACTTTTTATTTTTGA TATATATATTTTTATTTTTTAT A*T*A*T*A*T*C*G*T*A*T*A *T*A*C*C*C SEQ ID NO: 723 GGGTATATACTTTTTATTTTTGA TATATGTATTTTTATTTTTTAC A*T*A*T*A*T*C*G*T*A*T*A *T*A*C*C*C SEQ ID NO: 724 GGGTATATACTTTTTATTTTTGA TCATGTATTTTTTATTTTTATA C*A*T*G*A*T*C*G*T*A*T*A *T*A*C*C*C SEQ ID NO: 725 GGGTATATACTTTTTATTTTTGA TCATATATTTTTTATTTTTATA T*A*T*G*A*T*C*G*T*A*T*A *T*A*C*C*C SEQ ID NO: 726 GGATGTACACTTTTTATTTTTTA TATATATATTTTTATTTTTTAT A*T*A*T*A*T*A*G*T*G*T*A *C*A*T*C*C SEQ ID NO: 727 GGATGTACACTTTTTATTTTTGA TATATATATTTTTATTTTTTAT A*T*A*T*A*T*C*G*T*G*T*A *C*A*T*C*C SEQ ID NO: 728 GGATGTACACTTTTTATTTTTGA TATATGTATTTTTATTTTTTAC A*T*A*T*A*T*C*G*T*G*T*A *C*A*T*C*C SEQ ID NO: 729 GGATGTACACTTTTTATTTTTGA TCATGTATTTTTTATTTTTATA C*A*T*G*A*T*C*G*T*G*T*A *C*A*T*C*C SEQ ID NO: 730 GGATGTACACTTTTTATTTTTGA TCATATATTTTTTATTTTTATA T*A*T*G*A*T*C*G*T*G*T*A *C*A*T*C*C SEQ ID NO: 731 GTATATACTTTTTATTTTTTATA TATATATATATTTTTTATTTTT ATAT*A*T*A*T*A*T*A*T*A* T*A*G*T*A*T*ATAC SEQ ID NO: 732 GTATATACTTTTTATTTTTGATA TATATATATATTTTTTATTTTT ATAT*A*T*A*T*A*T*A*T*A* T*C*G*T*A*T*ATAC SEQ ID NO: 733 GGATATACTTTTTATTTTTTATA TATATATATATTTTTTATTTTT ATAT*A*T*A*T*A*T*A*T*A* T*A*G*T*A*T*ATCC SEQ ID NO: 734 GGATATACTTTTTATTTTTGATA TATATATATATTTTTTATTTTT ATAT*A*T*A*T*A*T*A*T*A* T*C*G*T*A*T*ATCC SEQ ID NO: 735 GGTGATACTTTTTATTTTTTATA TATATATATATTTTTTATTTTT ATAT*A*T*A*T*A*T*A*T*A* T*A*G*T*A*T*CACC SEQ ID NO: 736 GGTGATACTTTTTATTTTTGATA TATATATATATTTTTTATTTTT ATAT*A*T*A*T*A*T*A*T*A* T*C*G*T*A*T*CACC SEQ ID NO: 737 GGTGATCCTTTTTATTTTTTATA TATATATATATTTTTTATTTTT ATAT*A*T*A*T*A*T*A*T*A* T*A*G*G*A*T*CACC SEQ ID NO: 738 GGTGATCCTTTTTATTTTTGATA TATATATATATTTTTTATTTTT ATAT*A*T*A*T*A*T*A*T*A* T*C*G*G*A*T*CACC SEQ ID NO: 739 GATATATCACTTTTTATTTTTTA TATATATATATATTTTTTATTT TTATAT*A*T*A*T*A*T*A*T* A*T*A*G*T*G*A*TATATC SEQ ID NO: 740 GTATATACATTTTTTATTTTTGA TATATATATATATTTTTTATTT TTATAT*A*T*A*T*A*T*A*T* A*T*C*A*T*G*T*ATATAC SEQ ID NO: 741 GGATATACACTTTTTATTTTTTA TATATATATATATTTTTTATTT TTATAT*A*T*A*T*A*T*A*T* A*T*A*G*T*G*T*ATATCC SEQ ID NO: 742 GGATATACATTTTTTATTTTTGA TATATATATATATTTTTTATTT TTATAT*A*T*A*T*A*T*A*T* A*T*C*A*T*G*T*ATATCC SEQ ID NO: 743 GGGTATATACTTTTTATTTTTTA TATATATATATATTTTTTATTT TTATAT*A*T*A*T*A*T*A*T* A*T*A*G*T*A*T*ATACCC SEQ ID NO: 744 GGATATACACTTTTTATTTTTGA TATATATATATATTTTTTATTT TTATAT*A*T*A*T*A*T*A*T* A*T*C*G*T*G*T*ATATCC SEQ ID NO: 745 GGGTATATACTTTTTATTTTTGA TATATATATATATTTTTTATTT TTATAT*A*T*A*T*A*T*A*T* A*T*C*G*T*A*T*ATACCC SEQ ID NO: 746 GTATATACTTTTTATTTTTTATA TATATATATATTTTTATTTTTT ATA*T*A*T*A*T*A*T*A*T*A *G*T*A*T*A*TAC SEQ ID NO: 747 GTATATACTTTTTATTTTTGATA TATATATATATTTTTATTTTTT ATA*T*A*T*A*T*A*T*A*T*C *G*T*A*T*A*TAC SEQ ID NO: 748 GTATATACTTTTTATTTTTGATA TATGTATATATTTTTATTTTTT ATA*T*A*C*A*T*A*T*A*T*C *G*T*A*T*A*TAC SEQ ID NO: 749 GGATATACTTTTTATTTTTTATA TATATATATATTTTTATTTTTT ATA*T*A*T*A*T*A*T*A*T*A *G*T*A*T*A*TCC SEQ ID NO: 750 GGATATACTTTTTATTTTTGATA TATATATATATTTTTATTTTTT ATA*T*A*T*A*T*A*T*A*T*C *G*T*A*T*A*TCC SEQ ID NO: 751 GGATATACTTTTTATTTTTGATA TATGTATATATTTTTATTTTTT ATA*T*A*C*A*T*A*T*A*T*C *G*T*A*T*A*TCC SEQ ID NO: 752 GGTGATACTTTTTATTTTTTATA TATATATATATTTTTATTTTTT ATA*T*A*T*A*T*A*T*A*T*A *G*T*A*T*C*ACC SEQ ID NO: 753 GGTGATACTTTTTATTTTTGATA TATATATATATTTTTATTTTTT ATA*T*A*T*A*T*A*T*A*T*C *G*T*A*T*C*ACC SEQ ID NO: 754 GGTGATACTTTTTATTTTTGATA TATGTATATATTTTTATTTTTT ATA*T*A*C*A*T*A*T*A*T*C *G*T*A*T*C*ACC SEQ ID NO: 755 GGTGATCCTTTTTATTTTTTATA TATATATATATTTTTATTTTTT ATA*T*A*T*A*T*A*T*A*T*A *G*G*A*T*C*ACC SEQ ID NO: 756 GGTGATCCTTTTTATTTTTGATA TATATATATATTTTTATTTTTT ATA*T*A*T*A*T*A*T*A*T*C *G*G*A*T*C*ACC SEQ ID NO: 757 GGTGATCCTTTTTATTTTTGATA TATGTATATATTTTTATTTTTT ATA*T*A*C*A*T*A*T*A*T*C *G*G*A*T*C*ACC SEQ ID NO: 758 GATATATCACTTTTTATTTTTTA TATATATATATATTTTTATTTT TTATA*T*A*T*A*T*A*T*A*T *A*G*T*G*A*T*ATATC SEQ ID NO: 759 GTATATACATTTTTTATTTTTGA TATATATATATATTTTTATTTT TTATA*T*A*T*A*T*A*T*A*T *C*A*T*G*T*A*TATAC SEQ ID NO: 760 GTATATACATTTTTTATTTTTGA TATATGTATATATTTTTATTTT TTATA*T*A*C*A*T*A*T*A*T *C*A*T*G*T*A*TATAC SEQ ID NO: 761 GGATATACACTTTTTATTTTTTA TATATATATATATTTTTATTTT TTATA*T*A*T*A*T*A*T*A*T *A*G*T*G*T*A*TATCC SEQ ID NO: 762 GGATATACATTTTTTATTTTTGA TATATATATATATTTTTATTTT TTATA*T*A*T*A*T*A*T*A*T *C*A*T*G*T*A*TATCC SEQ ID NO: 763 GGATATACATTTTTTATTTTTGA TATATGTATATATTTTTATTTT TTATA*T*A*C*A*T*A*T*A*T *C*A*T*G*T*A*TATCC SEQ ID NO: 764 GGGTATATACTTTTTATTTTTTA TATATATATATATTTTTATTTT TTATA*T*A*T*A*T*A*T*A*T *A*G*T*A*T*A*TACCC SEQ ID NO: 765 GGATATACACTTTTTATTTTTGA TATATATATATATTTTTATTTT TTATA*T*A*T*A*T*A*T*A*T *C*G*T*G*T*A*TATCC SEQ ID NO: 766 GGATATACACTTTTTATTTTTGA TATATGTATATATTTTTATTTT TTATA*T*A*C*A*T*A*T*A*T *C*G*T*G*T*A*TATCC SEQ ID NO: 767 GGGTATATACTTTTTATTTTTGA TATATATATATATTTTTATTTT TTATA*T*A*T*A*T*A*T*A*T *C*G*T*A*T*A*TACCC SEQ ID NO: 768 GGGTATATACTTTTTATTTTTGA TATATGTATATATTTTTATTTT TTATA*T*A*C*A*T*A*T*A*T *C*G*T*A*T*A*TACCC SEQ ID NO: 769 GATATATCACTTTTTATTTTTTA TATATATATATTTTTTATTTTT ATAT*A*T*A*T*A*T*A*T*A* G*T*G*A*T*A*TATC SEQ ID NO: 770 GTATATACATTTTTTATTTTTGA TATATATATATTTTTTATTTTT ATAT*A*T*A*T*A*T*A*T*C* A*T*G*T*A*T*ATAC SEQ ID NO: 771 GTATATACATTTTTTATTTTTGA TATATGTATATTTTTTATTTTT ATAT*A*C*A*T*A*T*A*T*C* A*T*G*T*A*T*ATAC SEQ ID NO: 772 GGATATACACTTTTTATTTTTTA TATATATATATTTTTTATTTTT ATAT*A*T*A*T*A*T*A*T*A* G*T*G*T*A*T*ATCC SEQ ID NO: 773 GGATATACATTTTTTATTTTTGA TATATATATATTTTTTATTTTT ATAT*A*T*A*T*A*T*A*T*C* A*T*G*T*A*T*ATCC SEQ ID NO: 774 GGATATACATTTTTTATTTTTGA TATATGTATATTTTTTATTTTT ATAT*A*C*A*T*A*T*A*T*C* A*T*G*T*A*T*ATCC SEQ ID NO: 775 GGGTATATACTTTTTATTTTTTA TATATATATATTTTTTATTTTT ATAT*A*T*A*T*A*T*A*T*A* G*T*A*T*A*T*ACCC SEQ ID NO: 776 GGATATACACTTTTTATTTTTGA TATATATATATTTTTTATTTTT ATAT*A*T*A*T*A*T*A*T*C* G*T*G*T*A*T*ATCC SEQ ID NO: 777 GGATATACACTTTTTATTTTTGA TATATGTATATTTTTTATTTTT ATAT*A*C*A*T*A*T*A*T*C* G*T*G*T*A*T*ATCC SEQ ID NO: 778 GGGTATATACTTTTTATTTTTGA TATATATATATTTTTTATTTTT ATAT*A*T*A*T*A*T*A*T*C* G*T*A*T*A*T*ACCC SEQ ID NO: 779 GGGTATATACTTTTTATTTTTGA TATATGTATATTTTTTATTTTT ATAT*A*C*A*T*A*T*A*T*C* G*T*A*T*A*T*ACCC SEQ ID NO: 780 GATATATCACTTTTTATTTTTTA TATATATATATTTTTATTTTTT ATA*T*A*T*A*T*A*T*A*G*T *G*A*T*A*T*ATC SEQ ID NO: 781 GTATATACATTTTTTATTTTTGA TATATATATATTTTTATTTTTT ATA*T*A*T*A*T*A*T*C*A*T *G*T*A*T*A*TAC SEQ ID NO: 782 GTATATACATTTTTTATTTTTGA TATATGTATATTTTTATTTTTT ATA*C*A*T*A*T*A*T*C*A*T *G*T*A*T*A*TAC SEQ ID NO: 783 GTATATACATTTTTTATTTTTGA TCATGTATATTTTTTATTTTTA TAT*A*C*A*T*G*A*T*C*A*T *G*T*A*T*A*TAC SEQ ID NO: 784 GTATATACATTTTTTATTTTTGA TCATATATGTTTTTTATTTTTA CAT*A*T*A*T*G*A*T*C*A*T *G*T*A*T*A*TAC SEQ ID NO: 785 GGATATACACTTTTTATTTTTTA TATATATATATTTTTATTTTTT ATA*T*A*T*A*T*A*T*A*G*T *G*T*A*T*A*TCC SEQ ID NO: 786 GGATATACATTTTTTATTTTTGA TATATATATATTTTTATTTTTT ATA*T*A*T*A*T*A*T*C*A*T *G*T*A*T*A*TCC SEQ ID NO: 787 GGATATACATTTTTTATTTTTGA TATATGTATATTTTTATTTTTT ATA*C*A*T*A*T*A*T*C*A*T *G*T*A*T*A*TCC SEQ ID NO: 788 GGATATACATTTTTTATTTTTGA TCATGTATATTTTTTATTTTTA TAT*A*C*A*T*G*A*T*C*A*T *G*T*A*T*A*TCC SEQ ID NO: 789 GGATATACATTTTTTATTTTTGA TCATATATGTTTTTTATTTTTA CAT*A*T*A*T*G*A*T*C*A*T *G*T*A*T*A*TCC SEQ ID NO: 790 GGGTATATACTTTTTATTTTTTA TATATATATATTTTTATTTTTT ATA*T*A*T*A*T*A*T*A*G*T *A*T*A*T*A*CCC SEQ ID NO: 791 GGATATACACTTTTTATTTTTGA TATATATATATTTTTATTTTTT ATA*T*A*T*A*T*A*T*C*G*T *G*T*A*T*A*TCC SEQ ID NO: 792 GGATATACACTTTTTATTTTTGA TATATGTATATTTTTATTTTTT ATA*C*A*T*A*T*A*T*C*G*T *G*T*A*T*A*TCC SEQ ID NO: 793 GGATATACACTTTTTATTTTTGA TCATGTATATTTTTTATTTTTA TAT*A*C*A*T*G*A*T*C*G*T *G*T*A*T*A*TCC SEQ ID NO: 794 GGATATACACTTTTTATTTTTGA TCATATATGTTTTTTATTTTTA CAT*A*T*A*T*G*A*T*C*G*T *G*T*A*T*A*TCC SEQ ID NO: 795 GGGTATATACTTTTTATTTTTGA TATATATATATTTTTATTTTTT ATA*T*A*T*A*T*A*T*C*G*T *A*T*A*T*A*CCC SEQ ID NO: 796 GGGTATATACTTTTTATTTTTGA TATATGTATATTTTTATTTTTT ATA*C*A*T*A*T*A*T*C*G*T *A*T*A*T*A*CCC SEQ ID NO: 797 GGGTATATACTTTTTATTTTTGA TCATGTATATTTTTTATTTTTA TAT*A*C*A*T*G*A*T*C*G*T *A*T*A*T*A*CCC SEQ ID NO: 798 GGGTATATACTTTTTATTTTTGA TCATATATGTTTTTTATTTTTA CAT*A*T*A*T*G*A*T*C*G*T *A*T*A*T*A*CCC SEQ ID NO: 799 GTACATATATTTTTTTATTTTTG ATATATATATTTTTATTTTTTA TA*T*A*T*A*T*C*A*A*T*A* T*A*T*G*T*AC SEQ ID NO: 800 GTACATATATTTTTTTATTTTTG ATATATGTATTTTTATTTTTTA CA*T*A*T*A*T*C*A*A*T*A* T*A*T*G*T*AC SEQ ID NO: 801 GTACATATATTTTTTTATTTTTG ATCATGTATTTTTTATTTTTAT AC*A*T*G*A*T*C*A*A*T*A* T*A*T*G*T*AC SEQ ID NO: 802 GTACATATATTTTTTTATTTTTG ATCATATATTTTTTATTTTTAT AT*A*T*G*A*T*C*A*A*T*A* T*A*T*G*T*AC SEQ ID NO: 803 GATGTATATACTTTTTATTTTTT ATATATATATTTTTATTTTTTA TA*T*A*T*A*T*A*G*T*A*T* A*T*A*C*A*TC SEQ ID NO: 804 GGTACATATATTTTTTATTTTTG ATATATATATTTTTATTTTTTA TA*T*A*T*A*T*C*A*T*A*T* A*T*G*T*A*CC SEQ ID NO: 805 GGTACATATATTTTTTATTTTTG ATATATGTATTTTTATTTTTTA CA*T*A*T*A*T*C*A*T*A*T* A*T*G*T*A*CC SEQ ID NO: 806 GGTACATATATTTTTTATTTTTG ATCATGTATTTTTTATTTTTAT AC*A*T*G*A*T*C*A*T*A*T* A*T*G*T*A*CC SEQ ID NO: 807 GGTACATATATTTTTTATTTTTG ATCATATATTTTTTATTTTTAT AT*A*T*G*A*T*C*A*T*A*T* A*T*G*T*A*CC SEQ ID NO: 808 CGATCATATATTTTTTTATTTTT GATATATATATTTTTATTTTTT ATA*T*A*T*A*T*C*A*A*T*A *T*A*T*G*A*TCG SEQ ID NO: 809 CGATCATATATTTTTTTATTTTT GATATATGTATTTTTATTTTTT ACA*T*A*T*A*T*C*A*A*T*A *T*A*T*G*A*TCG SEQ ID NO: 810 CGATCATATATTTTTTTATTTTT GATCATGTATTTTTTATTTTTA TAC*A*T*G*A*T*C*A*A*T*A *T*A*T*G*A*TCG SEQ ID NO: 811 CGATCATATATTTTTTTATTTTT GATCATATATTTTTTATTTTTA TAT*A*T*G*A*T*C*A*A*T*A *T*A*T*G*A*TCG SEQ ID NO: 812 GATACTTTTTATTTTTTATAAAT ATATATATTTTTTATTTTTATA T*A*T*A*T*A*T*A*T*A*T*A *G*T*A*T*C SEQ ID NO: 813 GACACTTTTTATTTTTTATAAAT ATATATATTTTTTATTTTTATA T*A*T*A*T*A*T*A*T*A*T*A *G*T*G*T*C SEQ ID NO: 814 GATACTTTTTATTTTTGATAAAT ATATATATTTTTTATTTTTATA T*A*T*A*T*A*T*A*T*A*T*C *G*T*A*T*C SEQ ID NO: 815 GATACTTTTTATTTTTGATAAAT GTATATATTTTTTATTTTTATA T*A*T*A*C*A*T*A*T*A*T*C *G*T*A*T*C SEQ ID NO: 816 GATACTTTTTATTTTTGATGATG TATATATATTTTTATTTTTTAT A*T*A*T*A*C*A*T*G*A*T*C *G*T*A*T*C SEQ ID NO: 817 GATACTTTTTATTTTTGATGATA TATGTACTTTTTTATTTTTAGT A*C*A*T*A*T*A*T*G*A*T*C *G*T*A*T*C SEQ ID NO: 818 GGATCTTTTTATTTTTTATAAAT ATATATATTTTTTATTTTTATA T*A*T*A*T*A*T*A*T*A*T*A *G*A*T*C*C SEQ ID NO: 819 GACACTTTTTATTTTTGATAAAT ATATATATTTTTTATTTTTATA T*A*T*A*T*A*T*A*T*A*T*C *G*T*G*T*C SEQ ID NO: 820 GACACTTTTTATTTTTGATAAAT GTATATATTTTTTATTTTTATA T*A*T*A*C*A*T*A*T*A*T*C *G*T*G*T*C SEQ ID NO: 821 GACACTTTTTATTTTTGATGATG TATATATATTTTTATTTTTTAT A*T*A*T*A*C*A*T*G*A*T*C *G*T*G*T*C SEQ ID NO: 822 GACACTTTTTATTTTTGATGATA TATGTACTTTTTTATTTTTAGT A*C*A*T*A*T*A*T*G*A*T*C *G*T*G*T*C SEQ ID NO: 823 GGATCTTTTTATTTTTGATAAAT ATATATATTTTTTATTTTTATA T*A*T*A*T*A*T*A*T*A*T*C *G*A*T*C*C SEQ ID NO: 824 GGATCTTTTTATTTTTGATAAAT GTATATATTTTTTATTTTTATA T*A*T*A*C*A*T*A*T*A*T*C *G*A*T*C*C SEQ ID NO: 825 GGATCTTTTTATTTTTGATGATG TATATATATTTTTATTTTTTAT A*T*A*T*A*C*A*T*G*A*T*C *G*A*T*C*C SEQ ID NO: 826 GGATCTTTTTATTTTTGATGATA TATGTACTTTTTTATTTTTAGT A*C*A*T*A*T*A*T*G*A*T*C *G*A*T*C*C SEQ ID NO: 827 GCGTCTTTTTATTTTTTATAAAT ATATATATTTTTTATTTTTATA T*A*T*A*T*A*T*A*T*A*T*A *G*A*C*G*C SEQ ID NO: 828 GCGTCTTTTTATTTTTGATAAAT ATATATATTTTTTATTTTTATA T*A*T*A*T*A*T*A*T*A*T*C *G*A*C*G*C SEQ ID NO: 829 GCGTCTTTTTATTTTTGATAAAT GTATATATTTTTTATTTTTATA T*A*T*A*C*A*T*A*T*A*T*C *G*A*C*G*C SEQ ID NO: 830 GCGTCTTTTTATTTTTGATGATG TATATATATTTTTATTTTTTAT A*T*A*T*A*C*A*T*G*A*T*C *G*A*C*G*C SEQ ID NO: 831 GCGTCTTTTTATTTTTGATGATA TATGTACTTTTTTATTTTTAGT A*C*A*T*A*T*A*T*G*A*T*C *G*A*C*G*C SEQ ID NO: 832 GTATACTTTTTATTTTTGATAAA TATATATATTTTTATTTTTTAT A*T*A*T*A*T*A*T*A*T*C*G *T*A*T*A*C SEQ ID NO: 833 GTATACTTTTTATTTTTGATAAA TGTATATATTTTTATTTTTTAT A*T*A*C*A*T*A*T*A*T*C*G *T*A*T*A*C SEQ ID NO: 834 GTATACTTTTTATTTTTGATGAT GTATATATTTTTTATTTTTATA T*A*T*A*C*A*T*G*A*T*C*G *T*A*T*A*C SEQ ID NO: 835 GTATACTTTTTATTTTTGATGAT ATATGTACTTTTTATTTTTGTA C*A*T*A*T*A*T*G*A*T*C*G *T*A*T*A*C SEQ ID NO: 836 GTGATCTTTTTATTTTTGATAAA TATATATATTTTTATTTTTTAT A*T*A*T*A*T*A*T*A*T*C*G *A*T*C*A*C SEQ ID NO: 837 GTGATCTTTTTATTTTTGATAAA TGTATATATTTTTATTTTTTAT A*T*A*C*A*T*A*T*A*T*C*G *A*T*C*A*C SEQ ID NO: 838 GTGATCTTTTTATTTTTGATGAT GTATATATTTTTTATTTTTATA T*A*T*A*C*A*T*G*A*T*C*G *A*T*C*A*C SEQ ID NO: 839 GTGATCTTTTTATTTTTGATGAT ATATGTACTTTTTATTTTTGTA C*A*T*A*T*A*T*G*A*T*C*G *A*T*C*A*C SEQ ID NO: 840 GGATACTTTTTATTTTTGATAAA TATATATATTTTTATTTTTTAT A*T*A*T*A*T*A*T*A*T*C*G *T*A*T*C*C SEQ ID NO: 841 GGATACTTTTTATTTTTGATAAA TGTATATATTTTTATTTTTTAT A*T*A*C*A*T*A*T*A*T*C*G *T*A*T*C*C SEQ ID NO: 842 GGATACTTTTTATTTTTGATGAT GTATATATTTTTTATTTTTATA T*A*T*A*C*A*T*G*A*T*C*G *T*A*T*C*C SEQ ID NO: 843 GGATACTTTTTATTTTTGATGAT ATATGTACTTTTTATTTTTGTA C*A*T*A*T*A*T*G*A*T*C*G *T*A*T*C*C SEQ ID NO: 844 GCGATCTTTTTATTTTTGATAAA TATATATATTTTTATTTTTTAT A*T*A*T*A*T*A*T*A*T*C*G *A*T*C*G*C SEQ ID NO: 845 GCGATCTTTTTATTTTTGATAAA TGTATATATTTTTATTTTTTAT A*T*A*C*A*T*A*T*A*T*C*G *A*T*C*G*C SEQ ID NO: 846 GCGATCTTTTTATTTTTGATGAT GTATATATTTTTTATTTTTATA T*A*T*A*C*A*T*G*A*T*C*G *A*T*C*G*C SEQ ID NO: 847 GCGATCTTTTTATTTTTGATGAT ATATGTACTTTTTATTTTTGTA C*A*T*A*T*A*T*G*A*T*C*G *A*T*C*G*C SEQ ID NO: 848 GATATACTTTTTATTTTTTATAA ATATATATTTTTTATTTTTATA T*A*T*A*T*A*T*A*T*A*G*T *A*T*A*T*C SEQ ID NO: 849 GATATATTTTTTATTTTTGATAA ATGTATATTTTTTATTTTTATA T*A*C*A*T*A*T*A*T*C*A*T *A*T*A*T*C SEQ ID NO: 850 GATATATTTTTTATTTTTGATGA TGTATATATTTTTATTTTTTAT A*T*A*C*A*T*G*A*T*C*A*T *A*T*A*T*C SEQ ID NO: 851 GATATATTTTTTATTTTTGATGA TATATGTATTTTTATTTTTTAC A*T*A*T*A*T*G*A*T*C*A*T *A*T*A*T*C SEQ ID NO: 852 GTGATACTTTTTATTTTTTATAA ATATATATTTTTTATTTTTATA T*A*T*A*T*A*T*A*T*A*G*T *A*T*C*A*C SEQ ID NO: 853 GATATACTTTTTATTTTTGATAA ATATATATTTTTTATTTTTATA T*A*T*A*T*A*T*A*T*C*G*T *A*T*A*T*C SEQ ID NO: 854 GATATACTTTTTATTTTTGATGA TGTATATATTTTTATTTTTTAT A*T*A*C*A*T*G*A*T*C*G*T *A*T*A*T*C SEQ ID NO: 855 GATATACTTTTTATTTTTGATGA TATATGTATTTTTATTTTTTAC A*T*A*T*A*T*G*A*T*C*G*T *A*T*A*T*C SEQ ID NO: 856 GGTATACTTTTTATTTTTTATAA ATATATATTTTTTATTTTTATA T*A*T*A*T*A*T*A*T*A*G*T *A*T*A*C*C SEQ ID NO: 857 GTGATACTTTTTATTTTTGATAA ATATATATTTTTTATTTTTATA T*A*T*A*T*A*T*A*T*C*G*T *A*T*C*A*C SEQ ID NO: 858 GTGATACTTTTTATTTTTGATAA ATGTATATTTTTTATTTTTATA T*A*C*A*T*A*T*A*T*C*G*T *A*T*C*A*C SEQ ID NO: 859 GTGATACTTTTTATTTTTGATGA TGTATATATTTTTATTTTTTAT A*T*A*C*A*T*G*A*T*C*G*T *A*T*C*A*C SEQ ID NO: 860 GTGATACTTTTTATTTTTGATGA TATATGTATTTTTATTTTTTAC A*T*A*T*A*T*G*A*T*C*G*T *A*T*C*A*C SEQ ID NO: 861 GGTATACTTTTTATTTTTGATAA ATATATATTTTTTATTTTTATA T*A*T*A*T*A*T*A*T*C*G*T *A*T*A*C*C SEQ ID NO: 862 GGTATACTTTTTATTTTTGATAA ATGTATATTTTTTATTTTTATA T*A*C*A*T*A*T*A*T*C*G*T *A*T*A*C*C SEQ ID NO: 863 GGTATACTTTTTATTTTTGATGA TGTATATATTTTTATTTTTTAT A*T*A*C*A*T*G*A*T*C*G*T *A*T*A*C*C SEQ ID NO: 864 GGTATACTTTTTATTTTTGATGA TATATGTATTTTTATTTTTTAC A*T*A*T*A*T*G*A*T*C*G*T *A*T*A*C*C SEQ ID NO: 865 GGTGTACTTTTTATTTTTTATAA ATATATATTTTTTATTTTTATA T*A*T*A*T*A*T*A*T*A*G*T *A*C*A*C*C SEQ ID NO: 866 GGTGTACTTTTTATTTTTGATAA ATATATATTTTTTATTTTTATA T*A*T*A*T*A*T*A*T*C*G*T *A*C*A*C*C SEQ ID NO: 867 GGTGTACTTTTTATTTTTGATAA ATGTATATTTTTTATTTTTATA T*A*C*A*T*A*T*A*T*C*G*T *A*C*A*C*C SEQ ID NO: 868 GGTGTACTTTTTATTTTTGATGA TGTATATATTTTTATTTTTTAT A*T*A*C*A*T*G*A*T*C*G*T *A*C*A*C*C SEQ ID NO: 869 GGTGTACTTTTTATTTTTGATGA TATATGTATTTTTATTTTTTAC A*T*A*T*A*T*G*A*T*C*G*T *A*C*A*C*C SEQ ID NO: 870 GTATATACTTTTTATTTTTGATA AATATATATTTTTATTTTTTAT A*T*A*T*A*T*A*T*C*G*T*A *T*A*T*A*C SEQ ID NO: 871 GTATATACTTTTTATTTTTGATA AATGTATATTTTTATTTTTTAT A*C*A*T*A*T*A*T*C*G*T*A *T*A*T*A*C SEQ ID NO: 872 GTATATACTTTTTATTTTTGATG ATGTATATTTTTTATTTTTATA T*A*C*A*T*G*A*T*C*G*T*A *T*A*T*A*C SEQ ID NO: 873 GTATATACTTTTTATTTTTGATG ATATATGTTTTTTATTTTTACA T*A*T*A*T*G*A*T*C*G*T*A *T*A*T*A*C SEQ ID NO: 874 GGATATACTTTTTATTTTTGATA AATATATATTTTTATTTTTTAT A*T*A*T*A*T*A*T*C*G*T*A *T*A*T*C*C SEQ ID NO: 875 GGATATACTTTTTATTTTTGATA AATGTATATTTTTATTTTTTAT A*C*A*T*A*T*A*T*C*G*T*A *T*A*T*C*C SEQ ID NO: 876 GGATATACTTTTTATTTTTGATG ATGTATATTTTTTATTTTTATA T*A*C*A*T*G*A*T*C*G*T*A *T*A*T*C*C SEQ ID NO: 877 GGATATACTTTTTATTTTTGATG ATATATGTTTTTTATTTTTACA T*A*T*A*T*G*A*T*C*G*T*A *T*A*T*C*C SEQ ID NO: 878 GGTGATACTTTTTATTTTTGATA AATATATATTTTTATTTTTTAT A*T*A*T*A*T*A*T*C*G*T*A *T*C*A*C*C SEQ ID NO: 879 GGTGATACTTTTTATTTTTGATA AATGTATATTTTTATTTTTTAT A*C*A*T*A*T*A*T*C*G*T*A *T*C*A*C*C SEQ ID NO: 880 GGTGATACTTTTTATTTTTGATG ATGTATATTTTTTATTTTTATA T*A*C*A*T*G*A*T*C*G*T*A *T*C*A*C*C SEQ ID NO: 881 GGTGATACTTTTTATTTTTGATG ATATATGTTTTTTATTTTTACA T*A*T*A*T*G*A*T*C*G*T*A *T*C*A*C*C SEQ ID NO: 882 GGTGATCCTTTTTATTTTTGATA AATATATATTTTTATTTTTTAT A*T*A*T*A*T*A*T*C*G*G*A *T*C*A*C*C SEQ ID NO: 883 GGTGATCCTTTTTATTTTTGATA AATGTATATTTTTATTTTTTAT A*C*A*T*A*T*A*T*C*G*G*A *T*C*A*C*C SEQ ID NO: 884 GGTGATCCTTTTTATTTTTGATG ATGTATATTTTTTATTTTTATA T*A*C*A*T*G*A*T*C*G*G*A *T*C*A*C*C SEQ ID NO: 885 GGTGATCCTTTTTATTTTTGATG ATATATGTTTTTTATTTTTACA T*A*T*A*T*G*A*T*C*G*G*A *T*C*A*C*C SEQ ID NO: 886 GTATATACATTTTTTATTTTTGA TAAATATATTTTTATTTTTTAT A*T*A*T*A*T*C*A*T*G*T*A *T*A*T*A*C SEQ ID NO: 887 GTATATACATTTTTTATTTTTGA TAAATGTATTTTTATTTTTTAC A*T*A*T*A*T*C*A*T*G*T*A *T*A*T*A*C SEQ ID NO: 888 GTATATACATTTTTTATTTTTGA TGATGTATTTTTTATTTTTATA C*A*T*G*A*T*C*A*T*G*T*A *T*A*T*A*C SEQ ID NO: 889 GTATATACATTTTTTATTTTTGA TGATATATTTTTTATTTTTATA T*A*T*G*A*T*C*A*T*G*T*A *T*A*T*A*C SEQ ID NO: 890 GGATATACATTTTTTATTTTTGA TAAATATATTTTTATTTTTTAT A*T*A*T*A*T*C*A*T*G*T*A *T*A*T*C*C SEQ ID NO: 891 GGATATACATTTTTTATTTTTGA TAAATGTATTTTTATTTTTTAC A*T*A*T*A*T*C*A*T*G*T*A *T*A*T*C*C SEQ ID NO: 892 GGATATACATTTTTTATTTTTGA TGATGTATTTTTTATTTTTATA C*A*T*G*A*T*C*A*T*G*T*A *T*A*T*C*C SEQ ID NO: 893 GGATATACATTTTTTATTTTTGA TGATATATTTTTTATTTTTATA T*A*T*G*A*T*C*A*T*G*T*A *T*A*T*C*C SEQ ID NO: 894 GGATATACACTTTTTATTTTTGA TAAATATATTTTTATTTTTTAT A*T*A*T*A*T*C*G*T*G*T*A *T*A*T*C*C SEQ ID NO: 895 GGATATACACTTTTTATTTTTGA TAAATGTATTTTTATTTTTTAC A*T*A*T*A*T*C*G*T*G*T*A *T*A*T*C*C SEQ ID NO: 896 GGATATACACTTTTTATTTTTGA TGATGTATTTTTTATTTTTATA C*A*T*G*A*T*C*G*T*G*T*A *T*A*T*C*C SEQ ID NO: 897 GGATATACACTTTTTATTTTTGA TGATATATTTTTTATTTTTATA T*A*T*G*A*T*C*G*T*G*T*A *T*A*T*C*C SEQ ID NO: 898 GGGTATATACTTTTTATTTTTGA TAAATATATTTTTATTTTTTAT A*T*A*T*A*T*C*G*T*A*T*A *T*A*C*C*C SEQ ID NO: 899 GGGTATATACTTTTTATTTTTGA TAAATGTATTTTTATTTTTTAC A*T*A*T*A*T*C*G*T*A*T*A *T*A*C*C*C SEQ ID NO: 900 GGGTATATACTTTTTATTTTTGA TGATGTATTTTTTATTTTTATA C*A*T*G*A*T*C*G*T*A*T*A *T*A*C*C*C SEQ ID NO: 901 GGGTATATACTTTTTATTTTTGA TGATATATTTTTTATTTTTATA T*A*T*G*A*T*C*G*T*A*T*A *T*A*C*C*C SEQ ID NO: 902 GGATGTACACTTTTTATTTTTGA TAAATATATTTTTATTTTTTAT A*T*A*T*A*T*C*G*T*G*T*A *C*A*T*C*C SEQ ID NO: 903 GGATGTACACTTTTTATTTTTGA TAAATGTATTTTTATTTTTTAC A*T*A*T*A*T*C*G*T*G*T*A *C*A*T*C*C SEQ ID NO: 904 GGATGTACACTTTTTATTTTTGA TGATGTATTTTTTATTTTTATA C*A*T*G*A*T*C*G*T*G*T*A *C*A*T*C*C SEQ ID NO: 905 GGATGTACACTTTTTATTTTTGA TGATATATTTTTTATTTTTATA T*A*T*G*A*T*C*G*T*G*T*A *C*A*T*C*C SEQ ID NO: 906 GTATATACTTTTTATTTTTTATA AATATATATATTTTTTATTTTT ATAT*A*T*A*T*A*T*A*T*A* T*A*G*T*A*T*ATAC SEQ ID NO: 907 GTATATACTTTTTATTTTTGATA AATATATATATTTTTTATTTTT ATAT*A*T*A*T*A*T*A*T*A* T*C*G*T*A*T*ATAC SEQ ID NO: 908 GTATATACTTTTTATTTTTGATA AATGTATATATTTTTTATTTTT ATAT*A*T*A*C*A*T*A*T*A* T*C*G*T*A*T*ATAC SEQ ID NO: 909 GTATATACTTTTTATTTTTGATG ATGTATATATATTTTTATTTTT TATA*T*A*T*A*C*A*T*G*A* T*C*G*T*A*T*ATAC SEQ ID NO: 910 GTATATACTTTTTATTTTTGATG ATATATGTACTTTTTTATTTTT AGTA*C*A*T*A*T*A*T*G*A* T*C*G*T*A*T*ATAC SEQ ID NO: 911 GGATATACTTTTTATTTTTTATA AATATATATATTTTTTATTTTT ATAT*A*T*A*T*A*T*A*T*A* T*A*G*T*A*T*ATCC SEQ ID NO: 912 GGATATACTTTTTATTTTTGATA AATATATATATTTTTTATTTTT ATAT*A*T*A*T*A*T*A*T*A* T*C*G*T*A*T*ATCC SEQ ID NO: 913 GGATATACTTTTTATTTTTGATA AATGTATATATTTTTTATTTTT ATAT*A*T*A*C*A*T*A*T*A* T*C*G*T*A*T*ATCC SEQ ID NO: 914 GGATATACTTTTTATTTTTGATG ATGTATATATATTTTTATTTTT TATA*T*A*T*A*C*A*T*G*A* T*C*G*T*A*T*ATCC SEQ ID NO: 915 GGATATACTTTTTATTTTTGATG ATATATGTACTTTTTTATTTTT AGTA*C*A*T*A*T*A*T*G*A* T*C*G*T*A*T*ATCC SEQ ID NO: 916 GGTGATACTTTTTATTTTTTATA AATATATATATTTTTTATTTTT ATAT*A*T*A*T*A*T*A*T*A* T*A*G*T*A*T*CACC SEQ ID NO: 917 GGTGATACTTTTTATTTTTGATA AATATATATATTTTTTATTTTT ATAT*A*T*A*T*A*T*A*T*A* T*C*G*T*A*T*CACC SEQ ID NO: 918 GGTGATACTTTTTATTTTTGATA AATGTATATATTTTTTATTTTT ATAT*A*T*A*C*A*T*A*T*A* T*C*G*T*A*T*CACC SEQ ID NO: 919 GGTGATACTTTTTATTTTTGATG ATGTATATATATTTTTATTTTT TATA*T*A*T*A*C*A*T*G*A* T*C*G*T*A*T*CACC SEQ ID NO: 920 GGTGATACTTTTTATTTTTGATG ATATATGTACTTTTTTATTTTT AGTA*C*A*T*A*T*A*T*G*A* T*C*G*T*A*T*CACC SEQ ID NO: 921 GGTGATCCTTTTTATTTTTTATA AATATATATATTTTTTATTTTT ATAT*A*T*A*T*A*T*A*T*A* T*A*G*G*A*T*CACC SEQ ID NO: 922 GGTGATCCTTTTTATTTTTGATA AATATATATATTTTTTATTTTT ATAT*A*T*A*T*A*T*A*T*A* T*C*G*G*A*T*CACC SEQ ID NO: 923 GGTGATCCTTTTTATTTTTGATA AATGTATATATTTTTTATTTTT ATAT*A*T*A*C*A*T*A*T*A* T*C*G*G*A*T*CACC SEQ ID NO: 924 GGTGATCCTTTTTATTTTTGATG ATGTATATATATTTTTATTTTT TATA*T*A*T*A*C*A*T*G*A* T*C*G*G*A*T*CACC SEQ ID NO: 925 GGTGATCCTTTTTATTTTTGATG ATATATGTACTTTTTTATTTTT AGTA*C*A*T*A*T*A*T*G*A* T*C*G*G*A*T*CACC SEQ ID NO: 926 GATATATCACTTTTTATTTTTTA TAAATATATATATTTTTTATTT TTATAT*A*T*A*T*A*T*A*T* A*T*A*G*T*G*A*TATATC SEQ ID NO: 927 GTATATACATTTTTTATTTTTGA TAAATATATATATTTTTTATTT TTATAT*A*T*A*T*A*T*A*T* A*T*C*A*T*G*T*ATATAC SEQ ID NO: 928 GTATATACATTTTTTATTTTTGA TAAATGTATATATTTTTTATTT TTATAT*A*T*A*C*A*T*A*T* A*T*C*A*T*G*T*ATATAC SEQ ID NO: 929 GTATATACATTTTTTATTTTTGA TGATGTATATATATTTTTATTT TTTATA*T*A*T*A*C*A*T*G* A*T*C*A*T*G*T*ATATAC SEQ ID NO: 930 GTATATACATTTTTTATTTTTGA TGATATATGTACTTTTTTATTT TTAGTA*C*A*T*A*T*A*T*G* A*T*C*A*T*G*T*ATATAC SEQ ID NO: 931 GGATATACACTTTTTATTTTTTA TAAATATATATATTTTTTATTT TTATAT*A*T*A*T*A*T*A*T* A*T*A*G*T*G*T*ATATCC SEQ ID NO: 932 GGATATACATTTTTTATTTTTGA TAAATATATATATTTTTTATTT TTATAT*A*T*A*T*A*T*A*T* A*T*C*A*T*G*T*ATATCC SEQ ID NO: 933 GGATATACATTTTTTATTTTTGA TAAATGTATATATTTTTTATTT TTATAT*A*T*A*C*A*T*A*T* A*T*C*A*T*G*T*ATATCC SEQ ID NO: 934 GGATATACATTTTTTATTTTTGA TGATGTATATATATTTTTATTT TTTATA*T*A*T*A*C*A*T*G* A*T*C*A*T*G*T*ATATCC SEQ ID NO: 935 GGATATACATTTTTTATTTTTGA TGATATATGTACTTTTTTATTT TTAGTA*C*A*T*A*T*A*T*G* A*T*C*A*T*G*T*ATATCC SEQ ID NO: 936 GGGTATATACTTTTTATTTTTTA TAAATATATATATTTTTTATTT TTATAT*A*T*A*T*A*T*A*T* A*T*A*G*T*A*T*ATACCC SEQ ID NO: 937 GGATATACACTTTTTATTTTTGA TAAATATATATATTTTTTATT TTTATAT*A*T*A*T*A*T*A*T *A*T*C*G*T*G*T*ATATCC SEQ ID NO: 938 GGATATACACTTTTTATTTTTGA TAAATGTATATATTTTTTATT TTTATAT*A*T*A*C*A*T*A*T *A*T*C*G*T*G*T*ATATCC SEQ ID NO: 939 GGATATACACTTTTTATTTTTGA TGATGTATATATATTTTTATT TTTTATA*T*A*T*A*C*A*T*G *A*T*C*G*T*G*T*ATATCC SEQ ID NO: 940 GGATATACACTTTTTATTTTTGA TGATATATGTACTTTTTTATTT TTAGTA*C*A*T*A*T*A*T*G* A*T*C*G*T*G*T*ATATCC SEQ ID NO: 941 GGGTATATACTTTTTATTTTTGA TAAATATATATATTTTTTATTT TTATAT*A*T*A*T*A*T*A*T* A*T*C*G*T*A*T*ATACCC SEQ ID NO: 942 GGGTATATACTTTTTATTTTTGA TAAATGTATATATTTTTTATTT TTATAT*A*T*A*C*A*T*A*T* A*T*C*G*T*A*T*ATACCC SEQ ID NO: 943 GGGTATATACTTTTTATTTTTGA TGATGTATATATATTTTTATTT TTTATA*T*A*T*A*C*A*T*G* A*T*C*G*T*A*T*ATACCC SEQ ID NO: 944 GGGTATATACTTTTTATTTTTGA TGATATATGTACTTTTTTATTT TTAGTA*C*A*T*A*T*A*T*G* A*T*C*G*T*A*T*ATACCC SEQ ID NO: 945 GTATATACTTTTTATTTTTGATA AATATATATATTTTTATTTTTT ATA*T*A*T*A*T*A*T*A*T*C *G*T*A*T*A*TAC SEQ ID NO: 946 GTATATACTTTTTATTTTTGATA AATGTATATATTTTTATTTTTT ATA*T*A*C*A*T*A*T*A*T*C *G*T*A*T*A*TAC SEQ ID NO: 947 GTATATACTTTTTATTTTTGATG ATGTATATATTTTTTATTTTTA TAT*A*T*A*C*A*T*G*A*T*C *G*T*A*T*A*TAC SEQ ID NO: 948 GTATATACTTTTTATTTTTGATG ATATATGTACTTTTTATTTTTG TAC*A*T*A*T*A*T*G*A*T*C *G*T*A*T*A*TAC SEQ ID NO: 949 GGATATACTTTTTATTTTTGATA AATATATATATTTTTATTTTTT ATA*T*A*T*A*T*A*T*A*T*C *G*T*A*T*A*TCC SEQ ID NO: 950 GGATATACTTTTTATTTTTGATA AATGTATATATTTTTATTTTTT ATA*T*A*C*A*T*A*T*A*T*C *G*T*A*T*A*TCC SEQ ID NO: 951 GGATATACTTTTTATTTTTGATG ATGTATATATTTTTTATTTTTA TAT*A*T*A*C*A*T*G*A*T*C *G*T*A*T*A*TCC SEQ ID NO: 952 GGATATACTTTTTATTTTTGATG ATATATGTACTTTTTATTTTTG TAC*A*T*A*T*A*T*G*A*T*C *G*T*A*T*A*TCC SEQ ID NO: 953 GGTGATACTTTTTATTTTTGATA AATATATATATTTTTATTTTTT ATA*T*A*T*A*T*A*T*A*T*C *G*T*A*T*C*ACC SEQ ID NO: 954 GGTGATACTTTTTATTTTTGATA AATGTATATATTTTTATTTTTT ATA*T*A*C*A*T*A*T*A*T*C *G*T*A*T*C*ACC SEQ ID NO: 955 GGTGATACTTTTTATTTTTGATG ATGTATATATTTTTTATTTTTA TAT*A*T*A*C*A*T*G*A*T*C *G*T*A*T*C*ACC SEQ ID NO: 956 GGTGATACTTTTTATTTTTGATG ATATATGTACTTTTTATTTTTG TAC*A*T*A*T*A*T*G*A*T*C *G*T*A*T*C*ACC SEQ ID NO: 957 GGTGATCCTTTTTATTTTTGATA AATATATATATTTTTATTTTTT ATA*T*A*T*A*T*A*T*A*T*C *G*G*A*T*C*ACC SEQ ID NO: 958 GGTGATCCTTTTTATTTTTGATA AATGTATATATTTTTATTTTTT ATA*T*A*C*A*T*A*T*A*T*C *G*G*A*T*C*ACC SEQ ID NO: 959 GGTGATCCTTTTTATTTTTGATG ATGTATATATTTTTTATTTTTA TAT*A*T*A*C*A*T*G*A*T*C *G*G*A*T*C*ACC SEQ ID NO: 960 GGTGATCCTTTTTATTTTTGATG ATATATGTACTTTTTATTTTTG TAC*A*T*A*T*A*T*G*A*T*C *G*G*A*T*C*ACC SEQ ID NO: 961 GTATATACATTTTTTATTTTTGA TAAATATATATATTTTTATTTT TTATA*T*A*T*A*T*A*T*A*T *C*A*T*G*T*A*TATAC SEQ ID NO: 962 GTATATACATTTTTTATTTTTGA TGATGTATATATTTTTTATTTT TATAT*A*T*A*C*A*T*G*A*T *C*A*T*G*T*A*TATAC SEQ ID NO: 963 GTATATACATTTTTTATTTTTGA TGATATATGTACTTTTTATTTT TGTAC*A*T*A*T*A*T*G*A*T *C*A*T*G*T*A*TATAC SEQ ID NO: 964 GGATATACATTTTTTATTTTTGA TAAATATATATATTTTTATTTT TTATA*T*A*T*A*T*A*T*A*T *C*A*T*G*T*A*TATCC SEQ ID NO: 965 GGATATACATTTTTTATTTTTGA TAAATGTATATATTTTTATTTT TTATA*T*A*C*A*T*A*T*A*T *C*A*T*G*T*A*TATCC SEQ ID NO: 966 GGATATACATTTTTTATTTTTGA TGATGTATATATTTTTTATTTT TATAT*A*T*A*C*A*T*G*A*T *C*A*T*G*T*A*TATCC SEQ ID NO: 967 GGATATACATTTTTTATTTTTGA TGATATATGTACTTTTTATTTT TGTAC*A*T*A*T*A*T*G*A*T *C*A*T*G*T*A*TATCC SEQ ID NO: 968 GGATATACACTTTTTATTTTTGA TAAATATATATATTTTTATTT TTTATA*T*A*T*A*T*A*T*A* T*C*G*T*G*T*A*TATCC SEQ ID NO: 969 GGATATACACTTTTTATTTTTGA TAAATGTATATATTTTTATTT TTTATA*T*A*C*A*T*A*T*A* T*C*G*T*G*T*A*TATCC SEQ ID NO: 970 GGATATACACTTTTTATTTTTGA TGATGTATATATTTTTTATTTT TATAT*A*T*A*C*A*T*G*A*T *C*G*T*G*T*A*TATCC SEQ ID NO: 971 GGATATACACTTTTTATTTTTGA TGATATATGTACTTTTTATTTT TGTAC*A*T*A*T*A*T*G*A*T *C*G*T*G*T*A*TATCC SEQ ID NO: 972 GGGTATATACTTTTTATTTTTGA TAAATATATATATTTTTATTTT TTATA*T*A*T*A*T*A*T*A*T *C*G*T*A*T*A*TACCC SEQ ID NO: 973 GGGTATATACTTTTTATTTTTGA TAAATGTATATATTTTTATTTT TTATA*T*A*C*A*T*A*T*A*T *C*G*T*A*T*A*TACCC SEQ ID NO: 974 GGGTATATACTTTTTATTTTTGA TGATGTATATATTTTTTATTTT TATAT*A*T*A*C*A*T*G*A*T *C*G*T*A*T*A*TACCC SEQ ID NO: 975 GGGTATATACTTTTTATTTTTGA TGATATATGTACTTTTTATTTT TGTAC*A*T*A*T*A*T*G*A*T *C*G*T*A*T*A*TACCC SEQ ID NO: 976 GATATATCACTTTTTATTTTTTA TAAATATATATTTTTTATTTTT ATAT*A*T*A*T*A*T*A*T*A* G*T*G*A*T*A*TATC SEQ ID NO: 977 GTATATACATTTTTTATTTTTGA TAAATATATATTTTTTATTTTT ATAT*A*T*A*T*A*T*A*T*C* A*T*G*T*A*T*ATAC SEQ ID NO: 978 GTATATACATTTTTTATTTTTGA TGATGTATATATTTTTATTTTT TATA*T*A*C*A*T*G*A*T*C* A*T*G*T*A*T*ATAC SEQ ID NO: 979 GTATATACATTTTTTATTTTTGA TGATATATGTATTTTTATTTTT TACA*T*A*T*A*T*G*A*T*C* A*T*G*T*A*T*ATAC SEQ ID NO: 980 GGATATACACTTTTTATTTTTTA TAAATATATATTTTTTATTTTT ATAT*A*T*A*T*A*T*A*T*A* G*T*G*T*A*T*ATCC SEQ ID NO: 981 GGATATACATTTTTTATTTTTGA TAAATATATATTTTTTATTTTT ATAT*A*T*A*T*A*T*A*T*C* A*T*G*T*A*T*ATCC SEQ ID NO: 982 GGATATACATTTTTTATTTTTGA TAAATGTATATTTTTTATTTTT ATAT*A*C*A*T*A*T*A*T*C* A*T*G*T*A*T*ATCC SEQ ID NO: 983 GGATATACATTTTTTATTTTTGA TGATGTATATATTTTTATTTTT TATA*T*A*C*A*T*G*A*T*C* A*T*G*T*A*T*ATCC SEQ ID NO: 984 GGATATACATTTTTTATTTTTGA TGATATATGTATTTTTATTTTT TACA*T*A*T*A*T*G*A*T*C* A*T*G*T*A*T*ATCC SEQ ID NO: 985 GGGTATATACTTTTTATTTTTTA TAAATATATATTTTTTATTTTT ATAT*A*T*A*T*A*T*A*T*A* G*T*A*T*A*T*ACCC SEQ ID NO: 986 GGATATACACTTTTTATTTTTGA TAAATATATATTTTTTATTTTT ATAT*A*T*A*T*A*T*A*T*C* G*T*G*T*A*T*ATCC SEQ ID NO: 987 GGATATACACTTTTTATTTTTGA TAAATGTATATTTTTTATTTTT ATAT*A*C*A*T*A*T*A*T*C* G*T*G*T*A*T*ATCC SEQ ID NO: 988 GGATATACACTTTTTATTTTTGA TGATGTATATATTTTTATTTTT TATA*T*A*C*A*T*G*A*T*C* G*T*G*T*A*T*ATCC SEQ ID NO: 989 GGATATACACTTTTTATTTTTGA TGATATATGTATTTTTATTTTT TACA*T*A*T*A*T*G*A*T*C* G*T*G*T*A*T*ATCC SEQ ID NO: 990 GGGTATATACTTTTTATTTTTGA TAAATATATATTTTTTATTTTT ATAT*A*T*A*T*A*T*A*T*C* G*T*A*T*A*T*ACCC SEQ ID NO: 991 GGGTATATACTTTTTATTTTTGA TAAATGTATATTTTTTATTTTT ATAT*A*C*A*T*A*T*A*T*C* G*T*A*T*A*T*ACCC SEQ ID NO: 992 GGGTATATACTTTTTATTTTTGA TGATGTATATATTTTTATTTTT TATA*T*A*C*A*T*G*A*T*C* G*T*A*T*A*T*ACCC SEQ ID NO: 993 GGGTATATACTTTTTATTTTTGA TGATATATGTATTTTTATTTTT TACA*T*A*T*A*T*G*A*T*C* G*T*A*T*A*T*ACCC SEQ ID NO: 994 GTATATACATTTTTTATTTTTGA TAAATATATATTTTTATTTTTT ATA*T*A*T*A*T*A*T*C*A*T *G*T*A*T*A*TAC SEQ ID NO: 995 GTATATACATTTTTTATTTTTGA TGATGTATATTTTTTATTTTTA TAT*A*C*A*T*G*A*T*C*A*T *G*T*A*T*A*TAC SEQ ID NO: 996 GTATATACATTTTTTATTTTTGA TGATATATGTTTTTTATTTTTA CAT*A*T*A*T*G*A*T*C*A*T *G*T*A*T*A*TAC SEQ ID NO: 997 GGATATACATTTTTTATTTTTGA TAAATATATATTTTTATTTTTT ATA*T*A*T*A*T*A*T*C*A*T *G*T*A*T*A*TCC SEQ ID NO: 998 GGATATACATTTTTTATTTTTGA TAAATGTATATTTTTATTTTTT ATA*C*A*T*A*T*A*T*C*A*T *G*T*A*T*A*TCC SEQ ID NO: 999 GGATATACATTTTTTATTTTTGA TGATGTATATTTTTTATTTTTA TAT*A*C*A*T*G*A*T*C*A*T *G*T*A*T*A*TCC SEQ ID NO: 1000 GGATATACATTTTTTATTTTTGA TGATATATGTTTTTTATTTTTA CAT*A*T*A*T*G*A*T*C*A*T *G*T*A*T*A*TCC SEQ ID NO: 1001 GGATATACACTTTTTATTTTTGA TAAATATATATTTTTATTTTTT ATA*T*A*T*A*T*A*T*C*G*T *G*T*A*T*A*TCC SEQ ID NO: 1002 GGATATACACTTTTTATTTTTGA TAAATGTATATTTTTATTTTTT ATA*C*A*T*A*T*A*T*C*G*T *G*T*A*T*A*TCC SEQ ID NO: 1003 GGATATACACTTTTTATTTTTGA TGATGTATATTTTTTATTTTTA TAT*A*C*A*T*G*A*T*C*G*T *G*T*A*T*A*TCC SEQ ID NO: 1004 GGATATACACTTTTTATTTTTGA TGATATATGTTTTTTATTTTTA CAT*A*T*A*T*G*A*T*C*G*T *G*T*A*T*A*TCC SEQ ID NO: 1005 GGGTATATACTTTTTATTTTTGA TAAATATATATTTTTATTTTTT ATA*T*A*T*A*T*A*T*C*G*T *A*T*A*T*A*CCC SEQ ID NO: 1006 GGGTATATACTTTTTATTTTTGA TAAATGTATATTTTTATTTTTT ATA*C*A*T*A*T*A*T*C*G*T *A*T*A*T*A*CCC SEQ ID NO: 1007 GGGTATATACTTTTTATTTTTGA TGATGTATATTTTTTATTTTTA TAT*A*C*A*T*G*A*T*C*G*T *A*T*A*T*A*CCC SEQ ID NO: 1008 GGGTATATACTTTTTATTTTTGA TGATATATGTTTTTTATTTTTA CAT*A*T*A*T*G*A*T*C*G*T *A*T*A*T*A*CCC SEQ ID NO: 1009 GTACATATATTTTTTTATTTTTG ATAAATATATTTTTATTTTTTA TA*T*A*T*A*T*C*A*A*T*A* T*A*T*G*T*AC SEQ ID NO: 1010 GTACATATATTTTTTTATTTTTG ATAAATGTATTTTTATTTTTTA CA*T*A*T*A*T*C*A*A*T*A* T*A*T*G*T*AC SEQ ID NO: 1011 GTACATATATTTTTTTATTTTTG ATGATGTATTTTTTATTTTTAT AC*A*T*G*A*T*C*A*A*T*A* T*A*T*G*T*AC SEQ ID NO: 1012 GTACATATATTTTTTTATTTTTG ATGATATATTTTTTATTTTTAT AT*A*T*G*A*T*C*A*A*T*A* T*A*T*G*T*AC SEQ ID NO: 1013 GGTACATATATTTTTTATTTTTG ATAAATATATTTTTATTTTTTA TA*T*A*T*A*T*C*A*T*A*T* A*T*G*T*A*CC SEQ ID NO: 1014 GGTACATATATTTTTTATTTTTG ATAAATGTATTTTTATTTTTTA CA*T*A*T*A*T*C*A*T*A*T* A*T*G*T*A*CC SEQ ID NO: 1015 GGTACATATATTTTTTATTTTTG ATGATGTATTTTTTATTTTTAT AC*A*T*G*A*T*C*A*T*A*T* A*T*G*T*A*CC SEQ ID NO: 1016 GGTACATATATTTTTTATTTTTG ATGATATATTTTTTATTTTTAT AT*A*T*G*A*T*C*A*T*A*T* A*T*G*T*A*CC SEQ ID NO: 1017 CGATCATATATTTTTTTATTTTT GATAAATATATTTTTATTTTTT ATA*T*A*T*A*T*C*A*A*T*A *T*A*T*G*A*TCG SEQ ID NO: 1018 CGATCATATATTTTTTTATTTTT GATAAATGTATTTTTATTTTTT ACA*T*A*T*A*T*C*A*A*T*A *T*A*T*G*A*TCG SEQ ID NO: 1019 CGATCATATATTTTTTTATTTTT GATGATGTATTTTTTATTTTTA TAC*A*T*G*A*T*C*A*A*T*A *T*A*T*G*A*TCG SEQ ID NO: 1020 CGATCATATATTTTTTTATTTTT GATGATATATTTTTTATTTTTA TAT*A*T*G*A*T*C*A*A*T*A *T*A*T*G*A*TCG SEQ ID NO: 1021 GTATATACTTTTTATTTTTGATG ATGTAAATATATTTTTATTTTT TATA*T*A*T*A*C*A*T*G*A* T*C*G*T*A*T*ATAC SEQ ID NO: 1022 GTATATACTTTTTATTTTTGATG ATATAAGTACTTTTTTATTTTT AGTA*C*A*T*A*T*A*T*G*A* T*C*G*T*A*T*ATAC SEQ ID NO: 1023 GGATATACTTTTTATTTTTGATG ATGTAAATATATTTTTATTTTT TATA*T*A*T*A*C*A*T*G*A* T*C*G*T*A*T*ATCC SEQ ID NO: 1024 GGATATACTTTTTATTTTTGATG ATATAAGTACTTTTTTATTTTT AGTA*C*A*T*A*T*A*T*G*A* T*C*G*T*A*T*ATCC SEQ ID NO: 1025 GGTGATACTTTTTATTTTTGATG ATGTAAATATATTTTTATTTTT TATA*T*A*T*A*C*A*T*G*A* T*C*G*T*A*T*CACC SEQ ID NO: 1026 GGTGATACTTTTTATTTTTGATG ATATAAGTACTTTTTTATTTTT AGTA*C*A*T*A*T*A*T*G*A* T*C*G*T*A*T*CACC SEQ ID NO: 1027 GGTGATCCTTTTTATTTTTGATG ATGTAAATATATTTTTATTTTT TATA*T*A*T*A*C*A*T*G*A* T*C*G*G*A*T*CACC SEQ ID NO: 1028 GGTGATCCTTTTTATTTTTGATG ATATAAGTACTTTTTTATTTTT AGTA*C*A*T*A*T*A*T*G*A* T*C*G*G*A*T*CACC SEQ ID NO: 1029 GTATATACATTTTTTATTTTTGA TGATGTAAATATATTTTTATTT TTTATA*T*A*T*A*C*A*T*G* A*T*C*A*T*G*T*ATATAC SEQ ID NO: 1030 GTATATACATTTTTTATTTTTGA TGATATAAGTACTTTTTTATTT TTAGTA*C*A*T*A*T*A*T*G* A*T*C*A*T*G*T*ATATAC SEQ ID NO: 1031 GGATATACATTTTTTATTTTTGA TAAATGTAAATATTTTTTATT TTTATAT*A*T*A*C*A*T*A*T *A*T*C*A*T*G*T*ATATCC SEQ ID NO: 1032 GGATATACATTTTTTATTTTTGA TGATGTAAATATATTTTTATT TTTTATA*T*A*T*A*C*A*T*G *A*T*C*A*T*G*T*ATATCC SEQ ID NO: 1033 GGATATACATTTTTTATTTTTGA TGATATAAGTACTTTTTTATT TTTAGTA*C*A*T*A*T*A*T*G *A*T*C*A*T*G*T*ATATCC SEQ ID NO: 1034 GGATATACACTTTTTATTTTTGA TGATGTAAATATATTTTTATT TTTTATA*T*A*T*A*C*A*T*G *A*T*C*G*T*G*T*ATATCC SEQ ID NO: 1035 GGATATACACTTTTTATTTTTGA TGATATAAGTACTTTTTTATT TTTAGTA*C*A*T*A*T*A*T*G *A*T*C*G*T*G*T*ATATCC SEQ ID NO: 1036 GGGTATATACTTTTTATTTTTGA TGATGTAAATATATTTTTATT TTTTATA*T*A*T*A*C*A*T*G *A*T*C*G*T*A*T*ATACCC SEQ ID NO: 1037 GGGTATATACTTTTTATTTTTGA TGATATAAGTACTTTTTTATT TTTAGTA*C*A*T*A*T*A*T*G *A*T*C*G*T*A*T*ATACCC SEQ ID NO: 1038 GTATATACTTTTTATTTTTGATG ATGTAAATATTTTTTATTTTTA TAT*A*T*A*C*A*T*G*A*T*C *G*T*A*T*A*TAC SEQ ID NO: 1039 GTATATACTTTTTATTTTTGATG ATATAAGTACTTTTTATTTTTG TAC*A*T*A*T*A*T*G*A*T*C *G*T*A*T*A*TAC SEQ ID NO: 1040 GGATATACTTTTTATTTTTGATG ATGTAAATATTTTTTATTTTTA TAT*A*T*A*C*A*T*G*A*T*C *G*T*A*T*A*TCC SEQ ID NO: 1041 GGATATACTTTTTATTTTTGATG ATATAAGTACTTTTTATTTTT GTAC*A*T*A*T*A*T*G*A*T* C*G*T*A*T*A*TCC SEQ ID NO: 1042 GGTGATACTTTTTATTTTTGATG ATGTAAATATTTTTTATTTTTA TAT*A*T*A*C*A*T*G*A*T*C *G*T*A*T*C*ACC SEQ ID NO: 1043 GGTGATACTTTTTATTTTTGATG ATATAAGTACTTTTTATTTTT GTAC*A*T*A*T*A*T*G*A*T* C*G*T*A*T*C*ACC SEQ ID NO: 1044 GGTGATCCTTTTTATTTTTGATG ATGTAAATATTTTTTATTTTTA TAT*A*T*A*C*A*T*G*A*T*C *G*G*A*T*C*ACC SEQ ID NO: 1045 GGTGATCCTTTTTATTTTTGATG ATATAAGTACTTTTTATTTTTG TAC*A*T*A*T*A*T*G*A*T*C *G*G*A*T*C*ACC SEQ ID NO: 1046 GTATATACATTTTTTATTTTTGA TGATGTAAATATTTTTTATTTT TATAT*A*T*A*C*A*T*G*A*T *C*A*T*G*T*A*TATAC SEQ ID NO: 1047 GTATATACATTTTTTATTTTTGA TGATATAAGTACTTTTTATTTT TGTAC*A*T*A*T*A*T*G*A*T *C*A*T*G*T*A*TATAC SEQ ID NO: 1048 GGATATACATTTTTTATTTTTGA TGATGTAAATATTTTTTATTTT TATAT*A*T*A*C*A*T*G*A*T *C*A*T*G*T*A*TATCC SEQ ID NO: 1049 GGATATACATTTTTTATTTTTGA TGATATAAGTACTTTTTATTT TTGTAC*A*T*A*T*A*T*G*A* T*C*A*T*G*T*A*TATCC SEQ ID NO: 1050 GGATATACACTTTTTATTTTTGA TGATGTAAATATTTTTTATTT TTATAT*A*T*A*C*A*T*G*A* T*C*G*T*G*T*A*TATCC SEQ ID NO: 1051 GGATATACACTTTTTATTTTTGA TGATATAAGTACTTTTTATTT TTGTAC*A*T*A*T*A*T*G*A* T*C*G*T*G*T*A*TATCC SEQ ID NO: 1052 GGGTATATACTTTTTATTTTTGA TGATGTAAATATTTTTTATTTT TATAT*A*T*A*C*A*T*G*A*T *C*G*T*A*T*A*TACCC SEQ ID NO: 1053 GGGTATATACTTTTTATTTTTGA TGATATAAGTACTTTTTATTT TTGTAC*A*T*A*T*A*T*G*A* T*C*G*T*A*T*A*TACCC SEQ ID NO: 1054 GTATATACATTTTTTATTTTTGA TGATATAAGTATTTTTATTTTT TACA*T*A*T*A*T*G*A*T*C* A*T*G*T*A*T*ATAC SEQ ID NO: 1055 GGATATACATTTTTTATTTTTGA TAAATGAATATTTTTTATTTTT ATAT*A*C*A*T*A*T*A*T*C* A*T*G*T*A*T*ATCC SEQ ID NO: 1056 GGATATACATTTTTTATTTTTGA TGATATAAGTATTTTTATTTTT TACA*T*A*T*A*T*G*A*T*C* A*T*G*T*A*T*ATCC SEQ ID NO: 1057 GGATATACACTTTTTATTTTTGA TAAATGAATATTTTTTATTTT TATAT*A*C*A*T*A*T*A*T*C *G*T*G*T*A*T*ATCC SEQ ID NO: 1058 GGATATACACTTTTTATTTTTGA TGATATAAGTATTTTTATTTT TTACA*T*A*T*A*T*G*A*T*C *G*T*G*T*A*T*ATCC SEQ ID NO: 1059 GGGTATATACTTTTTATTTTTGA TAAATGAATATTTTTTATTTTT ATAT*A*C*A*T*A*T*A*T*C* G*T*A*T*A*T*ACCC SEQ ID NO: 1060 GGGTATATACTTTTTATTTTTGA TGATATAAGTATTTTTATTTTT TACA*T*A*T*A*T*G*A*T*C* G*T*A*T*A*T*ACCC SEQ ID NO: 1061 GTATATACATTTTTTATTTTTGA TGATGAATATTTTTTATTTTTA TAT*A*C*A*T*G*A*T*C*A*T *G*T*A*T*A*TAC SEQ ID NO: 1062 GGATATACATTTTTTATTTTTGA TAAATGAATATTTTTATTTTTT ATA*C*A*T*A*T*A*T*C*A*T *G*T*A*T*A*TCC SEQ ID NO: 1063 GGATATACATTTTTTATTTTTGA TGATGAATATTTTTTATTTTTA TAT*A*C*A*T*G*A*T*C*A*T *G*T*A*T*A*TCC SEQ ID NO: 1064 GGATATACATTTTTTATTTTTGA TGATAAATGTTTTTTATTTTTA CAT*A*T*A*T*G*A*T*C*A*T *G*T*A*T*A*TCC SEQ ID NO: 1065 GGATATACACTTTTTATTTTTGA TGATGAATATTTTTTATTTTT ATAT*A*C*A*T*G*A*T*C*G* T*G*T*A*T*A*TCC SEQ ID NO: 1066 GGGTATATACTTTTTATTTTTGA TGATGAATATTTTTTATTTTTA TAT*A*C*A*T*G*A*T*C*G*T *A*T*A*T*A*CCC SEQ ID NO: 1067 GATACTTTTTATTTTTGATGATG TAAATATATTTTTATTTTTTAT A*T*A*T*A*C*A*T*G*A*T*C *G*T*A*T*C SEQ ID NO: 1068 GATACTTTTTATTTTTGATGATA TAAGTACTTTTTTATTTTTAGT A*C*A*T*A*T*A*T*G*A*T*C *G*T*A*T*C SEQ ID NO: 1069 GACACTTTTTATTTTTGATGATG TAAATATATTTTTATTTTTTAT A*T*A*T*A*C*A*T*G*A*T*C *G*T*G*T*C SEQ ID NO: 1070 GACACTTTTTATTTTTGATGATA TAAGTACTTTTTTATTTTTAGT A*C*A*T*A*T*A*T*G*A*T*C *G*T*G*T*C SEQ ID NO: 1071 GGATCTTTTTATTTTTGATGATG TAAATATATTTTTATTTTTTAT A*T*A*T*A*C*A*T*G*A*T*C *G*A*T*C*C SEQ ID NO: 1072 GGATCTTTTTATTTTTGATGATA TAAGTACTTTTTTATTTTTAGT A*C*A*T*A*T*A*T*G*A*T*C *G*A*T*C*C SEQ ID NO: 1073 GCGTCTTTTTATTTTTGATGATG TAAATATATTTTTATTTTTTAT A*T*A*T*A*C*A*T*G*A*T*C *G*A*C*G*C SEQ ID NO: 1074 GCGTCTTTTTATTTTTGATGATA TAAGTACTTTTTTATTTTTAGT A*C*A*T*A*T*A*T*G*A*T*C *G*A*C*G*C SEQ ID NO: 1075 GTATACTTTTTATTTTTGATGAT GTAAATATTTTTTATTTTTATA T*A*T*A*C*A*T*G*A*T*C*G *T*A*T*A*C SEQ ID NO: 1076 GTATACTTTTTATTTTTGATGAT ATAAGTACTTTTTATTTTTGTA C*A*T*A*T*A*T*G*A*T*C*G *T*A*T*A*C SEQ ID NO: 1077 GTGATCTTTTTATTTTTGATGAT GTAAATATTTTTTATTTTTATA T*A*T*A*C*A*T*G*A*T*C*G *A*T*C*A*C SEQ ID NO: 1078 GTGATCTTTTTATTTTTGATGAT ATAAGTACTTTTTATTTTTGTA C*A*T*A*T*A*T*G*A*T*C*G *A*T*C*A*C SEQ ID NO: 1079 GGATACTTTTTATTTTTGATGAT GTAAATATTTTTTATTTTTATA T*A*T*A*C*A*T*G*A*T*C*G *T*A*T*C*C SEQ ID NO: 1080 GGATACTTTTTATTTTTGATGAT ATAAGTACTTTTTATTTTTGT AC*A*T*A*T*A*T*G*A*T*C* G*T*A*T*C*C SEQ ID NO: 1081 GCGATCTTTTTATTTTTGATGAT GTAAATATTTTTTATTTTTATA T*A*T*A*C*A*T*G*A*T*C*G *A*T*C*G*C SEQ ID NO: 1082 GCGATCTTTTTATTTTTGATGAT ATAAGTACTTTTTATTTTTGTA C*A*T*A*T*A*T*G*A*T*C*G *A*T*C*G*C SEQ ID NO: 1083 GATATATTTTTTATTTTTGATGA TATAAGTATTTTTATTTTTTAC A*T*A*T*A*T*G*A*T*C*A*T *A*T*A*T*C SEQ ID NO: 1084 GATATACTTTTTATTTTTGATGA TATAAGTATTTTTATTTTTTAC A*T*A*T*A*T*G*A*T*C*G*T *A*T*A*T*C SEQ ID NO: 1085 GTGATACTTTTTATTTTTGATAA ATGAATATTTTTTATTTTTATA T*A*C*A*T*A*T*A*T*C*G*T *A*T*C*A*C SEQ ID NO: 1086 GTGATACTTTTTATTTTTGATGA TATAAGTATTTTTATTTTTTAC A*T*A*T*A*T*G*A*T*C*G*T *A*T*C*A*C SEQ ID NO: 1087 GGTATACTTTTTATTTTTGATAA ATGAATATTTTTTATTTTTATA T*A*C*A*T*A*T*A*T*C*G*T *A*T*A*C*C SEQ ID NO: 1088 GGTATACTTTTTATTTTTGATGA TATAAGTATTTTTATTTTTTAC A*T*A*T*A*T*G*A*T*C*G*T *A*T*A*C*C SEQ ID NO: 1089 GGTGTACTTTTTATTTTTGATAA ATGAATATTTTTTATTTTTATA T*A*C*A*T*A*T*A*T*C*G*T *A*C*A*C*C SEQ ID NO: 1090 GGTGTACTTTTTATTTTTGATGA TATAAGTATTTTTATTTTTTAC A*T*A*T*A*T*G*A*T*C*G*T *A*C*A*C*C SEQ ID NO: 1091 GTATATACTTTTTATTTTTGATG ATGAATATTTTTTATTTTTATA T*A*C*A*T*G*A*T*C*G*T*A *T*A*T*A*C SEQ ID NO: 1092 GTATATACTTTTTATTTTTGATG ATAAATGTTTTTTATTTTTACA T*A*T*A*T*G*A*T*C*G*T*A *T*A*T*A*C SEQ ID NO: 1093 GGATATACTTTTTATTTTTGATG ATGAATATTTTTTATTTTTATA T*A*C*A*T*G*A*T*C*G*T*A *T*A*T*C*C SEQ ID NO: 1094 GGTGATACTTTTTATTTTTGATG ATGAATATTTTTTATTTTTATA T*A*C*A*T*G*A*T*C*G*T*A *T*C*A*C*C SEQ ID NO: 1095 GGTGATACTTTTTATTTTTGATG ATAAATGTTTTTTATTTTTAC AT*A*T*A*T*G*A*T*C*G*T* A*T*C*A*C*C SEQ ID NO: 1096 GGTGATCCTTTTTATTTTTGATG ATGAATATTTTTTATTTTTATA T*A*C*A*T*G*A*T*C*G*G*A *T*C*A*C*C SEQ ID NO: 1097 GATACTTTTTATTTTTGATATAA ATATATAATTTTTATTTTTATA T*A*T*A*T*A*T*A*T*A*T*C *G*T*A*T*C SEQ ID NO: 1098 GATACTTTTTATTTTTGATAAAT GAATATATTTTTTATTTTTATA T*A*T*A*C*A*T*A*T*A*T*C *G*T*A*T*C SEQ ID NO: 1099 GACACTTTTTATTTTTGATATAA ATATATAATTTTTATTTTTAT AT*A*T*A*T*A*T*A*T*A*T* C*G*T*G*T*C SEQ ID NO: 1100 GACACTTTTTATTTTTGATAAAT GAATATATTTTTTATTTTTAT AT*A*T*A*C*A*T*A*T*A*T* C*G*T*G*T*C SEQ ID NO: 1101 GACACTTTTTATTTTTGATATAA GTAAATATTTTTTATTTTTAT AT*A*T*A*C*A*T*A*T*A*T* C*G*T*G*T*C SEQ ID NO: 1102 GGATCTTTTTATTTTTGATATAA ATATATAATTTTTATTTTTATA T*A*T*A*T*A*T*A*T*A*T*C *G*A*T*C*C SEQ ID NO: 1103 GGATCTTTTTATTTTTGATAAAT GAATATATTTTTTATTTTTATA T*A*T*A*C*A*T*A*T*A*T*C *G*A*T*C*C SEQ ID NO: 1104 GGATCTTTTTATTTTTGATATAA GTAAATATTTTTTATTTTTATA T*A*T*A*C*A*T*A*T*A*T*C *G*A*T*C*C SEQ ID NO: 1105 GCGTCTTTTTATTTTTGATATAA ATATATAATTTTTATTTTTATA T*A*T*A*T*A*T*A*T*A*T*C *G*A*C*G*C SEQ ID NO: 1106 GCGTCTTTTTATTTTTGATAAAT GAATATATTTTTTATTTTTATA T*A*T*A*C*A*T*A*T*A*T*C *G*A*C*G*C SEQ ID NO: 1107 GCGTCTTTTTATTTTTGATATAA GTAAATATTTTTTATTTTTATA T*A*T*A*C*A*T*A*T*A*T*C *G*A*C*G*C SEQ ID NO: 1108 GTATATACATTTTTTATTTTTGA TATAAATATATAATTTTTATTT TTATAT*A*T*A*T*A*T*A*T* A*T*C*A*T*G*T*ATATAC SEQ ID NO: 1109 GTATATACATTTTTTATTTTTGA TAAATGAATATATTTTTTATTT TTATAT*A*T*A*C*A*T*A*T* A*T*C*A*T*G*T*ATATAC SEQ ID NO: 1110 GTATATACATTTTTTATTTTTGA TATAAGTAAATATTTTTTATTT TTATAT*A*T*A*C*A*T*A*T* A*T*C*A*T*G*T*ATATAC SEQ ID NO: 1111 GGATATACATTTTTTATTTTTGA TATAAATATATAATTTTTATT TTTATAT*A*T*A*T*A*T*A*T *A*T*C*A*T*G*T*ATATCC SEQ ID NO: 1112 GGATATACACTTTTTATTTTTGA TATAAATATATAATTTTTATT TTTATAT*A*T*A*T*A*T*A*T *A*T*C*G*T*G*T*ATATCC SEQ ID NO: 1113 GGATATACACTTTTTATTTTTGA TAAATGAATATATTTTTTATT TTTATAT*A*T*A*C*A*T*A*T *A*T*C*G*T*G*T*ATATCC SEQ ID NO: 1114 GGATATACACTTTTTATTTTTGA TATAAGTAAATATTTTTTATT TTTATAT*A*T*A*C*A*T*A*T *A*T*C*G*T*G*T*ATATCC SEQ ID NO: 1115 GGGTATATACTTTTTATTTTTGA TATAAATATATAATTTTTATT TTTATAT*A*T*A*T*A*T*A*T *A*T*C*G*T*A*T*ATACCC SEQ ID NO: 1116 GGGTATATACTTTTTATTTTTGA TAAATGAATATATTTTTTATT TTTATAT*A*T*A*C*A*T*A*T *A*T*C*G*T*A*T*ATACCC SEQ ID NO: 1117 GGGTATATACTTTTTATTTTTGA TATAAGTAAATATTTTTTATT TTTATAT*A*T*A*C*A*T*A*T *A*T*C*G*T*A*T*ATACCC SEQ ID NO: 1118 GTATACTTTTTATTTTTTATAAA TATATATTTTTTTATTTTTTAT A*T*A*T*A*T*A*T*A*T*A*G *T*A*T*A*C SEQ ID NO: 1119 GTGATCTTTTTATTTTTTATAAA TATATATTTTTTTATTTTTTAT A*T*A*T*A*T*A*T*A*T*A*G *A*T*C*A*C SEQ ID NO: 1120 GTATACTTTTTATTTTTGATATA AATATATTTTTTTATTTTTTAT A*T*A*T*A*T*A*T*A*T*C*G *T*A*T*A*C SEQ ID NO: 1121 GTATACTTTTTATTTTTGATATA TAAATATTTTTTTATTTTTTAT A*T*A*T*A*T*A*T*A*T*C*G *T*A*T*A*C SEQ ID NO: 1122 GTATACTTTTTATTTTTGATAAA TGAATATATTTTTATTTTTTAT A*T*A*C*A*T*A*T*A*T*C*G *T*A*T*A*C SEQ ID NO: 1123 GGATACTTTTTATTTTTTATAAA TATATATTTTTTTATTTTTTAT A*T*A*T*A*T*A*T*A*T*A*G *T*A*T*C*C SEQ ID NO: 1124 GTGATCTTTTTATTTTTGATATA AATATATTTTTTTATTTTTTAT A*T*A*T*A*T*A*T*A*T*C*G *A*T*C*A*C SEQ ID NO: 1125 GTGATCTTTTTATTTTTGATATA TAAATATTTTTTTATTTTTTAT A*T*A*T*A*T*A*T*A*T*C*G *A*T*C*A*C SEQ ID NO: 1126 GTGATCTTTTTATTTTTGATAAA TGAATATATTTTTATTTTTTAT A*T*A*C*A*T*A*T*A*T*C*G *A*T*C*A*C SEQ ID NO: 1127 GGATACTTTTTATTTTTGATATA AATATATTTTTTTATTTTTTAT A*T*A*T*A*T*A*T*A*T*C*G *T*A*T*C*C SEQ ID NO: 1128 GGATACTTTTTATTTTTGATATA TAAATATTTTTTTATTTTTTAT A*T*A*T*A*T*A*T*A*T*C*G *T*A*T*C*C SEQ ID NO: 1129 GGATACTTTTTATTTTTGATAAA TGAATATATTTTTATTTTTTAT A*T*A*C*A*T*A*T*A*T*C*G *T*A*T*C*C SEQ ID NO: 1130 GCGATCTTTTTATTTTTTATAAA TATATATTTTTTTATTTTTTAT A*T*A*T*A*T*A*T*A*T*A*G *A*T*C*G*C SEQ ID NO: 1131 GCGATCTTTTTATTTTTGATATA AATATATTTTTTTATTTTTTAT A*T*A*T*A*T*A*T*A*T*C*G *A*T*C*G*C SEQ ID NO: 1132 GCGATCTTTTTATTTTTGATATA TAAATATTTTTTTATTTTTTAT A*T*A*T*A*T*A*T*A*T*C*G *A*T*C*G*C SEQ ID NO: 1133 GCGATCTTTTTATTTTTGATAAA TGAATATATTTTTATTTTTTAT A*T*A*C*A*T*A*T*A*T*C*G *A*T*C*G*C SEQ ID NO: 1134 GATATATCACTTTTTATTTTTTA TAAATATATATTTTTTTATTTT TTATA*T*A*T*A*T*A*T*A*T *A*G*T*G*A*T*ATATC SEQ ID NO: 1135 GTATATACATTTTTTATTTTTGA TATAAATATATTTTTTTATTTT TTATA*T*A*T*A*T*A*T*A*T *C*A*T*G*T*A*TATAC SEQ ID NO: 1136 GTATATACATTTTTTATTTTTGA TATATAAATATTTTTTTATTTT TTATA*T*A*T*A*T*A*T*A*T *C*A*T*G*T*A*TATAC SEQ ID NO: 1137 GGATATACACTTTTTATTTTTTA TAAATATATATTTTTTTATTTT TTATA*T*A*T*A*T*A*T*A*T *A*G*T*G*T*A*TATCC SEQ ID NO: 1138 GGATATACATTTTTTATTTTTGA TATAAATATATTTTTTTATTTT TTATA*T*A*T*A*T*A*T*A*T *C*A*T*G*T*A*TATCC SEQ ID NO: 1139 GGATATACATTTTTTATTTTTGA TATATAAATATTTTTTTATTTT TTATA*T*A*T*A*T*A*T*A*T *C*A*T*G*T*A*TATCC SEQ ID NO: 1140 GGATATACATTTTTTATTTTTGA TAAATGAATATATTTTTATTT TTTATA*T*A*C*A*T*A*T*A* T*C*A*T*G*T*A*TATCC SEQ ID NO: 1141 GGGTATATACTTTTTATTTTTTA TAAATATATATTTTTTTATTTT TTATA*T*A*T*A*T*A*T*A*T *A*G*T*A*T*A*TACCC SEQ ID NO: 1142 GGATATACACTTTTTATTTTTGA TATAAATATATTTTTTTATTTT TTATA*T*A*T*A*T*A*T*A*T *C*G*T*G*T*A*TATCC SEQ ID NO: 1143 GGATATACACTTTTTATTTTTGA TATATAAATATTTTTTTATTTT TTATA*T*A*T*A*T*A*T*A*T *C*G*T*G*T*A*TATCC SEQ ID NO: 1144 GGATATACACTTTTTATTTTTGA TAAATGAATATATTTTTATTT TTTATA*T*A*C*A*T*A*T*A* T*C*G*T*G*T*A*TATCC SEQ ID NO: 1145 GGGTATATACTTTTTATTTTTGA TATAAATATATTTTTTTATTTT TTATA*T*A*T*A*T*A*T*A*T *C*G*T*A*T*A*TACCC SEQ ID NO: 1146 GGGTATATACTTTTTATTTTTGA TATATAAATATTTTTTTATTTT TTATA*T*A*T*A*T*A*T*A*T *C*G*T*A*T*A*TACCC SEQ ID NO: 1147 GGGTATATACTTTTTATTTTTGA TAAATGAATATATTTTTATTT TTTATA*T*A*C*A*T*A*T*A* T*C*G*T*A*T*A*TACCC SEQ ID NO: 1148 GATATACTTTTTATTTTTGATAA ATATATAATTTTTATTTTTATA T*A*T*A*T*A*T*A*T*C*G*T *A*T*A*T*C SEQ ID NO: 1149 GTGATACTTTTTATTTTTGATAA ATATATAATTTTTATTTTTATA T*A*T*A*T*A*T*A*T*C*G*T *A*T*C*A*C SEQ ID NO: 1150 GGTATACTTTTTATTTTTGATAA ATATATAATTTTTATTTTTATA T*A*T*A*T*A*T*A*T*C*G*T *A*T*A*C*C SEQ ID NO: 1151 GGTGTACTTTTTATTTTTGATAA ATATATAATTTTTATTTTTATA T*A*T*A*T*A*T*A*T*C*G*T *A*C*A*C*C SEQ ID NO: 1152 GTATATACATTTTTTATTTTTGA TAAATATATAATTTTTATTTTT ATAT*A*T*A*T*A*T*A*T*C* A*T*G*T*A*T*ATAC SEQ ID NO: 1153 GGATATACATTTTTTATTTTTGA TAAATATATAATTTTTATTTTT ATAT*A*T*A*T*A*T*A*T*C* A*T*G*T*A*T*ATCC SEQ ID NO: 1154 GGATATACACTTTTTATTTTTGA TAAATATATAATTTTTATTTT TATAT*A*T*A*T*A*T*A*T*C *G*T*G*T*A*T*ATCC SEQ ID NO: 1155 GGGTATATACTTTTTATTTTTGA TAAATATATAATTTTTATTTTT ATAT*A*T*A*T*A*T*A*T*C* G*T*A*T*A*T*ACCC SEQ ID NO: 1156 GTATATACTTTTTATTTTTGATA AATATATTTTTTTATTTTTTAT A*T*A*T*A*T*A*T*C*G*T*A *T*A*T*A*C SEQ ID NO: 1157 GTATATACTTTTTATTTTTGATA AATGTATTTTTTTATTTTTTAT A*C*A*T*A*T*A*T*C*G*T*A *T*A*T*A*C SEQ ID NO: 1158 GGATATACTTTTTATTTTTGATA AATATATTTTTTTATTTTTTAT A*T*A*T*A*T*A*T*C*G*T*A *T*A*T*C*C SEQ ID NO: 1159 GGATATACTTTTTATTTTTGATA AATGTATTTTTTTATTTTTTAT A*C*A*T*A*T*A*T*C*G*T*A *T*A*T*C*C SEQ ID NO: 1160 GGATATACTTTTTATTTTTGATG ATAAATGTTTTTTATTTTTAC AT*A*T*A*T*G*A*T*C*G*T* A*T*A*T*C*C SEQ ID NO: 1161 GGTGATACTTTTTATTTTTGATA AATATATTTTTTTATTTTTTAT A*T*A*T*A*T*A*T*C*G*T*A *T*C*A*C*C SEQ ID NO: 1162 GGTGATACTTTTTATTTTTGATA AATGTATTTTTTTATTTTTTAT A*C*A*T*A*T*A*T*C*G*T*A *T*C*A*C*C SEQ ID NO: 1163 GGTGATCCTTTTTATTTTTGATA AATATATTTTTTTATTTTTTAT A*T*A*T*A*T*A*T*C*G*G*A *T*C*A*C*C SEQ ID NO: 1164 GGTGATCCTTTTTATTTTTGATA AATGTATTTTTTTATTTTTTAT A*C*A*T*A*T*A*T*C*G*G*A *T*C*A*C*C SEQ ID NO: 1165 GGTGATCCTTTTTATTTTTGATG ATAAATGTTTTTTATTTTTACA T*A*T*A*T*G*A*T*C*G*G*A *T*C*A*C*C SEQ ID NO: 1166 GTATATACATTTTTTATTTTTGA TAAATATATTTTTTTATTTTTT ATA*T*A*T*A*T*A*T*C*A*T *G*T*A*T*A*TAC SEQ ID NO: 1167 GTATATACATTTTTTATTTTTGA TAAATGTATTTTTTTATTTTTT ATA*C*A*T*A*T*A*T*C*A*T *G*T*A*T*A*TAC SEQ ID NO: 1168 GTATATACATTTTTTATTTTTGA TGATAAATGTTTTTTATTTTTA CAT*A*T*A*T*G*A*T*C*A*T *G*T*A*T*A*TAC SEQ ID NO: 1169 GGATATACATTTTTTATTTTTGA TAAATATATTTTTTTATTTTTT ATA*T*A*T*A*T*A*T*C*A*T *G*T*A*T*A*TCC SEQ ID NO: 1170 GGATATACACTTTTTATTTTTGA TAAATATATTTTTTTATTTTTT ATA*T*A*T*A*T*A*T*C*G*T *G*T*A*T*A*TCC SEQ ID NO: 1171 GGATATACACTTTTTATTTTTGA TAAATGTATTTTTTTATTTTTT ATA*C*A*T*A*T*A*T*C*G*T *G*T*A*T*A*TCC SEQ ID NO: 1172 GGATATACACTTTTTATTTTTGA TGATAAATGTTTTTTATTTTT ACAT*A*T*A*T*G*A*T*C*G* T*G*T*A*T*A*TCC SEQ ID NO: 1173 GGGTATATACTTTTTATTTTTGA TAAATATATTTTTTTATTTTTT ATA*T*A*T*A*T*A*T*C*G*T *A*T*A*T*A*CCC SEQ ID NO: 1174 GGGTATATACTTTTTATTTTTGA TAAATGTATTTTTTTATTTTTT ATA*C*A*T*A*T*A*T*C*G*T *A*T*A*T*A*CCC SEQ ID NO: 1175 GGGTATATACTTTTTATTTTTGA TGATAAATGTTTTTTATTTTTA CAT*A*T*A*T*G*A*T*C*G*T *A*T*A*T*A*CCC SEQ ID NO: 1176 GTATATACATTTTTTATTTTTGA TAAATATTTTTTTATTTTTTAT A*T*A*T*A*T*C*A*T*G*T*A *T*A*T*A*C SEQ ID NO: 1177 GTATATACATTTTTTATTTTTGA TAAATGTTTTTTTATTTTTTAC A*T*A*T*A*T*C*A*T*G*T*A *T*A*T*A*C SEQ ID NO: 1178 GGATATACATTTTTTATTTTTGA TAAATATTTTTTTATTTTTTAT A*T*A*T*A*T*C*A*T*G*T*A *T*A*T*C*C SEQ ID NO: 1179 GGATATACATTTTTTATTTTTGA TAAATGTTTTTTTATTTTTTAC A*T*A*T*A*T*C*A*T*G*T*A *T*A*T*C*C SEQ ID NO: 1180 GGATATACATTTTTTATTTTTGA TGATGAATTTTTTATTTTTATA C*A*T*G*A*T*C*A*T*G*T*A *T*A*T*C*C SEQ ID NO: 1181 GGATATACACTTTTTATTTTTGA TAAATATTTTTTTATTTTTTAT A*T*A*T*A*T*C*G*T*G*T*A *T*A*T*C*C SEQ ID NO: 1182 GGATATACACTTTTTATTTTTGA TAAATGTTTTTTTATTTTTTAC A*T*A*T*A*T*C*G*T*G*T*A *T*A*T*C*C SEQ ID NO: 1183 GGGTATATACTTTTTATTTTTGA TAAATATTTTTTTATTTTTTAT A*T*A*T*A*T*C*G*T*A*T*A *T*A*C*C*C SEQ ID NO: 1184 GGGTATATACTTTTTATTTTTGA TAAATGTTTTTTTATTTTTTAC A*T*A*T*A*T*C*G*T*A*T*A *T*A*C*C*C SEQ ID NO: 1185 GGATGTACACTTTTTATTTTTGA TAAATATTTTTTTATTTTTTAT A*T*A*T*A*T*C*G*T*G*T*A *C*A*T*C*C SEQ ID NO: 1186 GGATGTACACTTTTTATTTTTGA TAAATGTTTTTTTATTTTTTAC A*T*A*T*A*T*C*G*T*G*T*A *C*A*T*C*C SEQ ID NO: 1187 GTACATATATTTTTTTATTTTTG ATAAATATTTTTTTATTTTTTA TA*T*A*T*A*T*C*A*A*T*A* T*A*T*G*T*AC SEQ ID NO: 1188 GTACATATATTTTTTTATTTTTG ATAAATGTTTTTTTATTTTTTA CA*T*A*T*A*T*C*A*A*T*A* T*A*T*G*T*AC SEQ ID NO: 1189 GGTACATATATTTTTTATTTTTG ATAAATATTTTTTTATTTTTTA TA*T*A*T*A*T*C*A*T*A*T* A*T*G*T*A*CC SEQ ID NO: 1190 GGTACATATATTTTTTATTTTTG ATAAATGTTTTTTTATTTTTTA CA*T*A*T*A*T*C*A*T*A*T* A*T*G*T*A*CC SEQ ID NO: 1191 CGATCATATATTTTTTTATTTTT GATAAATATTTTTTTATTTTTT ATA*T*A*T*A*T*C*A*A*T*A *T*A*T*G*A*TCG SEQ ID NO: 1192 CGATCATATATTTTTTTATTTTT GATAAATGTTTTTTTATTTTTT ACA*T*A*T*A*T*C*A*A*T*A *T*A*T*G*A*TCG SEQ ID NO: 1193 CGATCATATATTTTTTTATTTTT GATGATGAATTTTTTATTTTTA TAC*A*T*G*A*T*C*A*A*T*A *T*A*T*G*A*TCG SEQ ID NO: 1194 CGATCATATATTTTTTTATTTTT GATGATAAATTTTTTATTTTTA TAT*A*T*G*A*T*C*A*A*T*A *T*A*T*G*A*TCG SEQ ID NO: 1195 GTATATACTTTTTATTTTTGATA TAAATATATAATTTTTATTTTT ATAT*A*T*A*T*A*T*A*T*A* T*C*G*T*A*T*ATAC SEQ ID NO: 1196 GTATATACTTTTTATTTTTGATA AATGAATATATTTTTTATTTTT ATAT*A*T*A*C*A*T*A*T*A* T*C*G*T*A*T*ATAC SEQ ID NO: 1197 GGATATACTTTTTATTTTTGATA TAAATATATAATTTTTATTTTT ATAT*A*T*A*T*A*T*A*T*A* T*C*G*T*A*T*ATCC SEQ ID NO: 1198 GGATATACTTTTTATTTTTGATA AATGAATATATTTTTTATTTTT ATAT*A*T*A*C*A*T*A*T*A* T*C*G*T*A*T*ATCC SEQ ID NO: 1199 GGTGATACTTTTTATTTTTGATA TAAATATATAATTTTTATTTTT ATAT*A*T*A*T*A*T*A*T*A* T*C*G*T*A*T*CACC SEQ ID NO: 1200 GGTGATACTTTTTATTTTTGATA AATGAATATATTTTTTATTTTT ATAT*A*T*A*C*A*T*A*T*A* T*C*G*T*A*T*CACC SEQ ID NO: 1201 GGTGATCCTTTTTATTTTTGATA TAAATATATAATTTTTATTTTT ATAT*A*T*A*T*A*T*A*T*A* T*C*G*G*A*T*CACC SEQ ID NO: 1202 GGTGATCCTTTTTATTTTTGATA AATGAATATATTTTTTATTTTT ATAT*A*T*A*C*A*T*A*T*A* T*C*G*G*A*T*CACC SEQ ID NO: 1203 GTATATACTTTTTATTTTTGATA TAAGTAAATATTTTTTATTTTT ATAT*A*T*A*C*A*T*A*T*A* T*C*G*T*A*T*ATAC SEQ ID NO: 1204 GGATATACTTTTTATTTTTGATA TAAGTAAATATTTTTTATTTTT ATAT*A*T*A*C*A*T*A*T*A* T*C*G*T*A*T*ATCC SEQ ID NO: 1205 GGTGATACTTTTTATTTTTGATA TAAGTAAATATTTTTTATTTTT ATAT*A*T*A*C*A*T*A*T*A* T*C*G*T*A*T*CACC SEQ ID NO: 1206 GGTGATCCTTTTTATTTTTGATA TAAGTAAATATTTTTTATTTTT ATAT*A*T*A*C*A*T*A*T*A* T*C*G*G*A*T*CACC SEQ ID NO: 1207 GTATATACTTTTTATTTTTTATA AATATATATTTTTTTATTTTTT ATA*T*A*T*A*T*A*T*A*T*A *G*T*A*T*A*TAC SEQ ID NO: 1208 GTATATACTTTTTATTTTTGATA TAAATATATTTTTTTATTTTTT ATA*T*A*T*A*T*A*T*A*T*C *G*T*A*T*A*TAC SEQ ID NO: 1209 GTATATACTTTTTATTTTTGATA AATGAATATATTTTTATTTTTT ATA*T*A*C*A*T*A*T*A*T*C *G*T*A*T*A*TAC SEQ ID NO: 1210 GGATATACTTTTTATTTTTTATA AATATATATTTTTTTATTTTTT ATA*T*A*T*A*T*A*T*A*T*A *G*T*A*T*A*TCC SEQ ID NO: 1211 GGATATACTTTTTATTTTTGATA TAAATATATTTTTTTATTTTTT ATA*T*A*T*A*T*A*T*A*T*C *G*T*A*T*A*TCC SEQ ID NO: 1212 GGATATACTTTTTATTTTTGATA AATGAATATATTTTTATTTTTT ATA*T*A*C*A*T*A*T*A*T*C *G*T*A*T*A*TCC SEQ ID NO: 1213 GGTGATACTTTTTATTTTTTATA AATATATATTTTTTTATTTTTT ATA*T*A*T*A*T*A*T*A*T*A *G*T*A*T*C*ACC SEQ ID NO: 1214 GGTGATACTTTTTATTTTTGATA TAAATATATTTTTTTATTTTTT ATA*T*A*T*A*T*A*T*A*T*C *G*T*A*T*C*ACC SEQ ID NO: 1215 GGTGATACTTTTTATTTTTGATA AATGAATATATTTTTATTTTTT ATA*T*A*C*A*T*A*T*A*T*C *G*T*A*T*C*ACC SEQ ID NO: 1216 GGTGATCCTTTTTATTTTTTATA AATATATATTTTTTTATTTTTT ATA*T*A*T*A*T*A*T*A*T*A *G*G*A*T*C*ACC SEQ ID NO: 1217 GGTGATCCTTTTTATTTTTGATA TAAATATATTTTTTTATTTTTT ATA*T*A*T*A*T*A*T*A*T*C *G*G*A*T*C*ACC SEQ ID NO: 1218 GGTGATCCTTTTTATTTTTGATA AATGAATATATTTTTATTTTTT ATA*T*A*C*A*T*A*T*A*T*C *G*G*A*T*C*ACC SEQ ID NO: 1219 GTATATACTTTTTATTTTTGATA TATAAATATTTTTTTATTTTTT ATA*T*A*T*A*T*A*T*A*T*C *G*T*A*T*A*TAC SEQ ID NO: 1220 GGATATACTTTTTATTTTTGATA TATAAATATTTTTTTATTTTTT ATA*T*A*T*A*T*A*T*A*T*C *G*T*A*T*A*TCC SEQ ID NO: 1221 GGTGATACTTTTTATTTTTGATA TATAAATATTTTTTTATTTTTT ATA*T*A*T*A*T*A*T*A*T*C *G*T*A*T*C*ACC SEQ ID NO: 1222 GGTGATCCTTTTTATTTTTGATA TATAAATATTTTTTTATTTTTT ATA*T*A*T*A*T*A*T*A*T*C *G*G*A*T*C*ACC SEQ ID NO: 1223 GATACAAAAAAAAAAATATATAT ATATATATAAAAAAAAAAA ATAT*A*T*A*T*A*T*A*T*A* T*A*G*T*A*T*C SEQ ID NO: 1224 GACACAAAAAAAAAAAGATATAT ATATATATAAAAAAAAAAA ATAT*A*T*A*T*A*T*A*T*A* T*C*G*T*G*T*C SEQ ID NO: 1225 GATATACAAAAAAAAAAATATAT ATATATATAAAAAAAAAAA ATAT*A*T*A*T*A*T*A*T*A* G*T*A*T*A*T*C SEQ ID NO: 1226 GATATATAAAAAAAAAAAGATAT ATGTATATAAAAAAAAAAA ATAT*A*C*A*T*A*T*A*T*C* A*T*A*T*A*T*C SEQ ID NO: 1227 GATATACAAAAAAAAAAAGATAT ATATATATAAAAAAAAAAA ATAT*A*T*A*T*A*T*A*T*C* G*T*A*T*A*T*C SEQ ID NO: 1228 GGTATACAAAAAAAAAAATATAT ATATATATAAAAAAAAAAA ATAT*A*T*A*T*A*T*A*T*A* G*T*A*T*A*C*C SEQ ID NO: 1229 GATATATCACAAAAAAAAAAATA TATATATAAAAAAAAAAAA TATA*T*A*T*A*T*A*G*T*G* A*T*A*T*A*T*C SEQ ID NO: 1230 GTATATACATAAAAAAAAAAAGA TATATGTAAAAAAAAAAAA TACA*T*A*T*A*T*C*A*T*G* T*A*T*A*T*A*C SEQ ID NO: 1231 GGATATACATAAAAAAAAAAAGA TATATGTAAAAAAAAAAA ATACA*T*A*T*A*T*C*A*T*G *T*A*T*A*T*C*C SEQ ID NO: 1232 GGATATACATAAAAAAAAAAAGA TCATGTATAAAAAAAAAAA ATAC*A*T*G*A*T*C*A*T*G* T*A*T*A*T*C*C SEQ ID NO: 1233 GGGTATATACAAAAAAAAAAATA TATATATAAAAAAAAAAAA TATA*T*A*T*A*T*A*G*T*A* T*A*T*A*C*C*C SEQ ID NO: 1234 GTATATACAAAAAAAAAAATATA TATATATATATAAAAAAAA AAAATAT*A*T*A*T*A*T*A*T *A*T*A*G*T*A*T*ATAC SEQ ID NO: 1235 GTATATACAAAAAAAAAAAGATA TATATATATATAAAAAAAA AAAATAT*A*T*A*T*A*T*A*T *A*T*C*G*T*A*T*ATAC SEQ ID NO: 1236 GGATATACAAAAAAAAAAATATA TATATATATATAAAAAAAA AAAATAT*A*T*A*T*A*T*A*T *A*T*A*G*T*A*T*ATCC SEQ ID NO: 1237 GGATATACAAAAAAAAAAAGATA TATATATATATAAAAAAAA AAAATAT*A*T*A*T*A*T*A*T *A*T*C*G*T*A*T*ATCC SEQ ID NO: 1238 GTATATACAAAAAAAAAAATATA TATATATATAAAAAAAAAA AATATA*T*A*T*A*T*A*T*A* T*A*G*T*A*T*A*TAC SEQ ID NO: 1239 GTATATACAAAAAAAAAAAGATA TATATATATAAAAAAAAAA AATATA*T*A*T*A*T*A*T*A* T*C*G*T*A*T*A*TAC SEQ ID NO: 1240 GGATATACAAAAAAAAAAATATA TATATATATAAAAAAAAAA AATATA*T*A*T*A*T*A*T*A* T*A*G*T*A*T*A*TCC SEQ ID NO: 1241 GGATATACAAAAAAAAAAAGATA TATATATATAAAAAAAAAA AATATA*T*A*T*A*T*A*T*A* T*C*G*T*A*T*A*TCC SEQ ID NO: 1242 GATATATCACAAAAAAAAAAATA TATATATATAAAAAAAAAA AATATA*T*A*T*A*T*A*T*A* G*T*G*A*T*A*T*ATC SEQ ID NO: 1243 GGATATACATAAAAAAAAAAAGA TATATATATAAAAAAAAAA AATATA*T*A*T*A*T*A*T*C* A*T*G*T*A*T*A*TCC SEQ ID NO: 1244 GTACATATATTAAAAAAAAAAAG ATATATATAAAAAAAAAAA ATATA*T*A*T*A*T*C*A*A*T *A*T*A*T*G*T*AC SEQ ID NO: 1245 GATGTATATACAAAAAAAAAAAT ATATATATAAAAAAAAAAA ATATA*T*A*T*A*T*A*G*T*A *T*A*T*A*C*A*TC SEQ ID NO: 1246 CGATCATATATTAAAAAAAAAAA GATATATATAAAAAAAAAA AATATA*T*A*T*A*T*C*A*A* T*A*T*A*T*G*A*TCG SEQ ID NO: 1247 CGATCATATATTAAAAAAAAAAA GATATATGTAAAAAAAAAA AATACA*T*A*T*A*T*C*A*A* T*A*T*A*T*G*A*TCG SEQ ID NO: 1248 GATACAAAAAAAAAAATATAAAT ATATATATAAAAAAAAAAA ATAT*A*T*A*T*A*T*A*T*A* T*A*G*T*A*T*C SEQ ID NO: 1249 GGATCAAAAAAAAAAATATAAAT ATATATATAAAAAAAAAAA ATAT*A*T*A*T*A*T*A*T*A* T*A*G*A*T*C*C SEQ ID NO: 1250 GACACAAAAAAAAAAAGATAAAT ATATATATAAAAAAAAAA AATAT*A*T*A*T*A*T*A*T*A *T*C*G*T*G*T*C SEQ ID NO: 1251 GACACAAAAAAAAAAAGATGATG TATATATAAAAAAAAAAA ATATA*T*A*T*A*C*A*T*G*A *T*C*G*T*G*T*C SEQ ID NO: 1252 GCGTCAAAAAAAAAAAGATAAAT ATATATATAAAAAAAAAAA ATAT*A*T*A*T*A*T*A*T*A* T*C*G*A*C*G*C SEQ ID NO: 1253 GATATACAAAAAAAAAAATATAA ATATATATAAAAAAAAAAA ATAT*A*T*A*T*A*T*A*T*A* G*T*A*T*A*T*C SEQ ID NO: 1254 GTATATACATAAAAAAAAAAAGA TAAATGTAAAAAAAAAAA ATACA*T*A*T*A*T*C*A*T*G *T*A*T*A*T*A*C SEQ ID NO: 1255 GTATATACATAAAAAAAAAAAGA TGATATATAAAAAAAAAAA ATAT*A*T*G*A*T*C*A*T*G* T*A*T*A*T*A*C SEQ ID NO: 1256 GGATATACATAAAAAAAAAAAGA TAAATATAAAAAAAAAAA ATATA*T*A*T*A*T*C*A*T*G *T*A*T*A*T*C*C SEQ ID NO: 1257 GGATATACATAAAAAAAAAAAGA TGATATATAAAAAAAAAA AATAT*A*T*G*A*T*C*A*T*G *T*A*T*A*T*C*C SEQ ID NO: 1258 GTATATACAAAAAAAAAAATATA AATATATATATAAAAAAAA AAAATAT*A*T*A*T*A*T*A*T *A*T*A*G*T*A*T*ATAC SEQ ID NO: 1259 GTATATACAAAAAAAAAAAGATA AATATATATATAAAAAAAA AAAATAT*A*T*A*T*A*T*A*T *A*T*C*G*T*A*T*ATAC SEQ ID NO: 1260 GGATATACAAAAAAAAAAATATA AATATATATATAAAAAAAA AAAATAT*A*T*A*T*A*T*A*T *A*T*A*G*T*A*T*ATCC SEQ ID NO: 1261 GGATATACAAAAAAAAAAAGATA AATATATATATAAAAAAAA AAAATAT*A*T*A*T*A*T*A*T *A*T*C*G*T*A*T*ATCC SEQ ID NO: 1262 GTATATACAAAAAAAAAAAGATA AATGTATATAAAAAAAAAA AATATA*T*A*C*A*T*A*T*A* T*C*G*T*A*T*A*TAC SEQ ID NO: 1263 GGATATACAAAAAAAAAAAGATA AATGTATATAAAAAAAAA AAATATA*T*A*C*A*T*A*T*A *T*C*G*T*A*T*A*TCC SEQ ID NO: 1264 GGTGATACAAAAAAAAAAAGATG ATGTATATATAAAAAAAAA AAATAT*A*T*A*C*A*T*G*A* T*C*G*T*A*T*C*ACC SEQ ID NO: 1265 GATATATCACAAAAAAAAAAATA TAAATATATATAAAAAAAA AAAATAT*A*T*A*T*A*T*A*T *A*G*T*G*A*T*A*TATC SEQ ID NO: 1266 GTATATACATAAAAAAAAAAAGA TAAATATATAAAAAAAAAA AATATA*T*A*T*A*T*A*T*C* A*T*G*T*A*T*A*TAC SEQ ID NO: 1267 GTATATACATAAAAAAAAAAAGA TGATATATGTAAAAAAAAA AAACAT*A*T*A*T*G*A*T*C* A*T*G*T*A*T*A*TAC SEQ ID NO: 1268 GGATATACATAAAAAAAAAAAGA TAAATATATAAAAAAAAA AAATATA*T*A*T*A*T*A*T*C *A*T*G*T*A*T*A*TCC SEQ ID NO: 1269 GTACATATATTAAAAAAAAAAAG ATAAATATAAAAAAAAAAA ATATA*T*A*T*A*T*C*A*A*T *A*T*A*T*G*T*AC SEQ ID NO: 1270 GTACATATATTAAAAAAAAAAAG ATAAATGTAAAAAAAAAAA ATACA*T*A*T*A*T*C*A*A*T *A*T*A*T*G*T*AC SEQ ID NO: 1271 GTACATATATTAAAAAAAAAAAG ATGATATATAAAAAAAAAA AATAT*A*T*G*A*T*C*A*A*T *A*T*A*T*G*T*AC SEQ ID NO: 1272 GGATATACATAAAAAAAAAAAGA TGATGAATAAAAAAAAAA AATAC*A*T*G*A*T*C*A*T*G *T*A*T*A*T*C*C SEQ ID NO: 1273 GTATATACATAAAAAAAAAAAGA TAAATGTTAAAAAAAAAAA TACA*T*A*T*A*T*C*A*T*G* T*A*T*A*T*A*C SEQ ID NO: 1274 GATACAAAAAAAAAAAGATATAA ATATATAAAAAAAAAAAA AATAT*A*T*A*T*A*T*A*T*A *T*C*G*T*A*T*C SEQ ID NO: 1275 GATACAAAAAAAAAAAGATGATA TAAGTACTAAAAAAAAAA AAGTA*C*A*T*A*T*A*T*G*A *T*C*G*T*A*T*C SEQ ID NO: 1276 GACACAAAAAAAAAAAGATAAAT GAATATATAAAAAAAAAA AATAT*A*T*A*C*A*T*A*T*A *T*C*G*T*G*T*C SEQ ID NO: 1277 GGATATACAAAAAAAAAAAGATA TAAGTAAATATAAAAAAA AAAAATAT*A*T*A*C*A*T*A* T*A*T*C*G*T*A*T*ATCC SEQ ID NO: 1278 GGATATACAAAAAAAAAAAGATG ATATAAGTACTAAAAAAAA AAAAGTA*C*A*T*A*T*A*T*G *A*T*C*G*T*A*T*ATCC SEQ ID NO: 1279 GTATATACAAAAAAAAAAAGATA TAAGTAAATATAAAAAAAA AAAATAT*A*T*A*C*A*T*A*T *A*T*C*G*T*A*T*ATAC SEQ ID NO: 1280 GTATATACAAAAAAAAAAAGATG ATATAAGTACTAAAAAAAA AAAAGTA*C*A*T*A*T*A*T*G *A*T*C*G*T*A*T*ATAC SEQ ID NO: 1281 GGATATACAAAAAAAAAAAGATA AATGAATATAAAAAAAAA AAATATA*T*A*C*A*T*A*T*A *T*C*G*T*A*T*A*TCC SEQ ID NO: 1282 GTATATACAAAAAAAAAAAGATA AATGAATATAAAAAAAAA AAATATA*T*A*C*A*T*A*T*A *T*C*G*T*A*T*A*TAC SEQ ID NO: 1283 GTATATACAAAAAAAAAAAGATG ATATAAGTACAAAAAAAAA AAGTAC*A*T*A*T*A*T*G*A* T*C*G*T*A*T*A*TAC SEQ ID NO: 1284 GTATATACAAAAAAAAAAATATA AATATATATTAAAAAAAAA AATATA*T*A*T*A*T*A*T*A* T*A*G*T*A*T*A*TAC SEQ ID NO: 1285 GTATATACATAAAAAAAAAAAGA TGATGTAAATATAAAAAAA AAAAATAT*A*T*A*C*A*T*G* A*T*C*A*T*G*T*A*TATAC SEQ ID NO: 1286 GATATACAAAAAAAAAAAGATAA ATATATAAAAAAAAAAAA AATAT*A*T*A*T*A*T*A*T*C *G*T*A*T*A*T*C SEQ ID NO: 1287 GTGATACAAAAAAAAAAAGATAA ATATATAAAAAAAAAAAA AATAT*A*T*A*T*A*T*A*T*C *G*T*A*T*C*A*C SEQ ID NO: 1288 GGTATACAAAAAAAAAAAGATAA ATATATAAAAAAAAAAAA AATAT*A*T*A*T*A*T*A*T*C *G*T*A*T*A*C*C SEQ ID NO: 1289 GGATATACATAAAAAAAAAAAGA TAAATGAATAAAAAAAAA AAATATA*C*A*T*A*T*A*T*C *A*T*G*T*A*T*A*TCC SEQ ID NO: 1290 GTATATACATAAAAAAAAAAAGA TAAATGTATTAAAAAAAAA AATATA*C*A*T*A*T*A*T*C* A*T*G*T*A*T*A*TAC SEQ ID NO: 1291 GTATATACATAAAAAAAAAAAGA TGATAAATGTAAAAAAAAA AAACAT*A*T*A*T*G*A*T*C* A*T*G*T*A*T*A*TAC SEQ ID NO: 1292 TATATATTATTTTATTTTAATCG AGTCTTTTTGACTCGATATAC AATATATA SEQ ID NO: 1293 GATATATTATTTTATTTTAATCG AGTCTTTTTGACTCGATATAC AATATATC SEQ ID NO: 1294 GATATATCATTTTATTTTAATCG AGTCTTTTTGACTCGATATAC GATATATC SEQ ID NO: 1295 GATATGTCATTTTATTTTAATCG AGTCTTTTTGACTCGATATAC GACATATC SEQ ID NO: 1296 GTGATGTCATTTTATTTTAATCG AGTCTTTTTGACTCGATATAC GACATCAC SEQ ID NO: 1297 TATATATTATTTTATTTTATGCG AGTCTTTTTGACTCGCAGCCC AATATATA SEQ ID NO: 1298 GATATATTATTTTATTTTATGCG AGTCTTTTTGACTCGCAGCCC AATATATC SEQ ID NO: 1299 GATATATCATTTTATTTTATGCG AGTCTTTTTGACTCGCAGCCC GATATATC SEQ ID NO: 1300 GATATGTCATTTTATTTTATGCG AGTCTTTTTGACTCGCAGCCC GACATATC SEQ ID NO: 1301 GTGATGTCATTTTATTTTATGCG AGTCTTTTTGACTCGCAGCCC GACATCAC SEQ ID NO: 1302 TATATATTATTTTATTTTAGTAT ATCGGACTCGATATACAATAT ATA SEQ ID NO: 1303 GATATATTATTTTATTTTAGTAT ATCGGACTCGATATACAATAT ATC SEQ ID NO: 1304 GATATATCATTTTATTTTAGTAT ATCGGACTCGATATACGATAT ATC SEQ ID NO: 1305 GATATGTCATTTTATTTTAGTAT ATCGGACTCGATATACGACAT ATC SEQ ID NO: 1306 GTGATGTCATTTTATTTTAGTAT ATCGGACTCGATATACGACAT CAC SEQ ID NO: 1307 TATATATTATTTTATTTTAGGGC TGCGGACTCGCAGCCCAATAT ATA SEQ ID NO: 1308 GATATATTATTTTATTTTAGGGC TGCGGACTCGCAGCCCAATA TATC SEQ ID NO: 1309 GATATATCATTTTATTTTAGGGC TGCGGACTCGCAGCCCGATA TATC SEQ ID NO: 1310 GATATGTCATTTTATTTTAGGGC TGCGGACTCGCAGCCCGACA TATC SEQ ID NO: 1311 GTGATGTCATTTTATTTTAGGGC TGCGGACTCGCAGCCCGACA TCAC SEQ ID NO: 1312 TATATATATTATTACTATATGGA CTCGCATATAGATATATA SEQ ID NO: 1313 GATATATATTATTACTATATGGA CTCGCATATAGATATATC SEQ ID NO: 1314 GATATACATTATTACTATATGGA CTCGCATATAGGTATATC SEQ ID NO: 1315 GATATCCATTATTACTATATGGA CTCGCATATAGGGATATC SEQ ID NO: 1316 GTGATACATTATTACTATATGGA CTCGCATATAGGTATCAC SEQ ID NO: 1317 TATATATTTTATTTCGGGCTGGA CTCGCAGCCCGATATATA SEQ ID NO: 1318 GATATATATTATTACGGGCTGGA CTCGCAGCCCGATATATC SEQ ID NO: 1319 GATATACATTATTACGGGCTGGA CTCGCAGCCCGGTATATC SEQ ID NO: 1320 GATATCCATTATTACGGGCTGGA CTCGCAGCCCGGGATATC SEQ ID NO: 1321 GTGATACATTATTACGGGCTGGA CTCGCAGCCCGGTATCAC SEQ ID NO: 1322 TATATATTTTATTTCTATATGTT TATTTCGAGTCTTTTGACTCGC ATATAGATATATA SEQ ID NO: 1323 GATATATATTATTACTATATGAT TATTACGAGTCTTTTGACTCG CATATAGATATATC SEQ ID NO: 1324 GATATACATTATTACTATATGAT TATTACGAGTCTTTTGACTCG CATATAGGTATATC SEQ ID NO: 1325 GATATCCATTATTACTATATGAT TATTACGAGTCTTTTGACTCG CATATAGGGATATC SEQ ID NO: 1326 GTGATACATTATTACTATATGAT TATTACGAGTCTTTTGACTCG CATATAGGTATCAC SEQ ID NO: 1327 TATATATATTATTACGGGCTGAT TATTACGAGTCTTTTGACTCG CAGCCCGATATATA SEQ ID NO: 1328 GATATATATTATTACGGGCTGAT TATTACGAGTCTTTTGACTCG CAGCCCGATATATC SEQ ID NO: 1329 GATATACATTATTACGGGCTGAT TATTACGAGTCTTTTGACTC GCAGCCCGGTATATC SEQ ID NO: 1330 GATATCCATTATTACGGGCTGAT TATTACGAGTCTTTTGACTCG CAGCCCGGGATATC SEQ ID NO: 1331 GTGATACATTATTACGGGCTGAT TATTACGAGTCTTTTGACTC GCAGCCCGGTATCAC SEQ ID NO: 1332 TATATATTTATTTCATATCGACT CGCAGATATGTATATA SEQ ID NO: 1333 GATATCATTATTACATATCGACT CGCAGATATGGATATC SEQ ID NO: 1334 GTGATCATTATTACATATCGACT CGCAGATATGGATCAC SEQ ID NO: 1335 GTGTGCATTATTACATATCGACT CGCAGATATGGCACAC SEQ ID NO: 1336 GATATCATTATTACCGGGCGACT CGCAGCCCGGGATATC SEQ ID NO: 1337 GTGATCATTATTACCGGGCGACT CGCAGCCCGGGATCAC SEQ ID NO: 1338 GTGTGCATTATTACCGGGCGACT CGCAGCCCGGGCACAC SEQ ID NO: 1339 TATATATTTATTTCATATCTTTA TTTTGCGAGTCTTTTGACTCGC AGATATGTATATA SEQ ID NO: 1340 GATATCATTATTACATATCATTA TTATGCGAGTCTTTTGACTCG CAGATATGGATATC SEQ ID NO: 1341 GTGATCATTATTACATATCATTA TTATGCGAGTCTTTTGACTCG CAGATATGGATCAC SEQ ID NO: 1342 GTGTGCATTATTACATATCATTA TTATGCGAGTCTTTTGACTCG CAGATATGGCACAC SEQ ID NO: 1343 GATATCATTATTACCGGGCATTA TTATGCGAGTCTTTTGACTCG CAGCCCGGGATATC SEQ ID NO: 1344 GTGATCATTATTACCGGGCATTA TTATGCGAGTCTTTTGACTCG CAGCCCGGGATCAC SEQ ID NO: 1345 GTGTGCATTATTACCGGGCATTA TTATGCGAGTCTTTTGACTCG CAGCCCGGGCACAC SEQ ID NO: 1346 GTATGATTATTACACAGGACTCG CAGCCTGTGCATAC SEQ ID NO: 1347 GTGTGATTATTACACAGGACTCG CAGCCTGTGCACAC SEQ ID NO: 1348 GTATGATTATTACCCGGGACTCG CAGCCCGGGCATAC SEQ ID NO: 1349 GTGTGATTATTACCCGGGACTCG CAGCCCGGGCACAC SEQ ID NO: 1350 GTATGATTATTACACAGATTATT AGCTGCATTATTAGAGTCTTT TGACTCGCAGCCTGTGCATAC SEQ ID NO: 1351 GTGTGATTATTACACAGATTATT AGCTGCATTATTAGAGTCTTT TGACTCGCAGCCTGTGCACAC SEQ ID NO: 1352 TATATATTATTACCCGGATTATT AGCTGCATTATTAGAGTCTTT TGACTCGCAGCCCGGGATATA SEQ ID NO: 1353 GATATATTATTACCCGGATTATT AGCTGCATTATTAGAGTCTTT TGACTCGCAGCCCGGGATATC SEQ ID NO: 1354 GTATGATTATTACCCGGATTATT AGCTGCATTATTAGAGTCTTT TGACTCGCAGCCCGGGCATAC SEQ ID NO: 1355 GTGTGATTATTACCCGGATTATT AGCTGCATTATTAGAGTCTTT TGACTCGCAGCCCGGGCACAC SEQ ID NO: 1356 GACTCGATATACAATATATAGCG CGCGCAATAAGCGCGCATT ATTAGCTATATAATTATTATTGT ATAT SEQ ID NO: 1357 GACTCGATATACAATATATCGCG CGCGCAATAAGCGCGCATTA TTAGCGATATAATTATTATTGTA TAT SEQ ID NO: 1358 GACTCGATATACGATATATCGCG CGCGCAATAAGCGCGCATTA TTAGCGATATAATTATTATCGTA TAT SEQ ID NO: 1359 GACTCGATATACGACATATCGCG CGCGCAATAAGCGCGCATT ATTAGCGATATGATTATTATCGT ATAT SEQ ID NO: 1360 GACTCGATATACGACATCACGCG CGCGCAATAAGCGCGCATT ATTAGCGTGATGATTATTATCGT ATAT SEQ ID NO: 1361 GACTCGCAGCCCAATATATAGCG CGCGCAATAAGCGCGCATT ATTAGCTATATAATTATTATTGG GCTG SEQ ID NO: 1362 GACTCGCAGCCCAATATATCGCG CGCGCAATAAGCGCGCATT ATTAGCGATATAATTATTATTGG GCTG SEQ ID NO: 1363 GACTCGCAGCCCGATATATCGCG CGCGCAATAAGCGCGCATT ATTAGCGATATAATTATTATCGG GCTG SEQ ID NO: 1364 GACTCGCAGCCCGACATATCGCG CGCGCAATAAGCGCGCATT ATTAGCGATATGATTATTATCGG GCTG SEQ ID NO: 1365 GACTCGCAGCCCGACATCACGCG CGCGCAATAAGCGCGCATT ATTAGCGTGATGATTATTATCGG GCTG SEQ ID NO: 1366 GACTCGATATACAATATATAGCG CGCGCAATAAGCGCGCATT ATTAGCTATATAATTATTATTGT ATATTATTAATCGAGTC SEQ ID NO: 1367 GACTCGATATACAATATATCGCG CGCGCAATAAGCGCGCATTA TTAGCGATATAATTATTATTGTA TATTATTAATCGAGTC SEQ ID NO: 1368 GACTCGATATACGATATATCGCG CGCGCAATAAGCGCGCATTA TTAGCGATATAATTATTATCGTA TATTATTAATCGAGTC SEQ ID NO: 1369 GACTCGATATACGACATATCGCG CGCGCAATAAGCGCGCATT ATTAGCGATATGATTATTATCGT ATATTATTAATCGAGTC SEQ ID NO: 1370 GACTCGATATACGACATCACGCG CGCGCAATAAGCGCGCATT ATTAGCGTGATGATTATTATCGT ATATTATTAATCGAGTC SEQ ID NO: 1371 GACTCGCAGCCCAATATATAGCG CGCGCAATAAGCGCGCATT ATTAGCTATATAATTATTATTGG GCATTATTATGCGAGTC SEQ ID NO: 1372 GACTCGCAGCCCAATATATCGCG CGCGCAATAAGCGCGCATT ATTAGCGATATAATTATTATTGG GCATTATTATGCGAGTC SEQ ID NO: 1373 GACTCGCAGCCCGATATATCGCG CGCGCAATAAGCGCGCATT ATTAGCGATATAATTATTATCGG GCATTATTATGCGAGTC SEQ ID NO: 1374 GACTCGCAGCCCGACATATCGCG CGCGCAATAAGCGCGCATT ATTAGCGATATGATTATTATCGG GCATTATTATGCGAGTC SEQ ID NO: 1375 GACTCGCAGCCCGACATCACGCG CGCGCAATAAGCGCGCATT ATTAGCGTGATGATTATTATCGG GCATTATTATGCGAGTC SEQ ID NO: 1376 GACTCGCATATAGATATATAGCG CGCGCAATAAGCGCGCATT ATTAGCTATATATTATTAATCTA TA SEQ ID NO: 1377 GACTCGCATATAGATATATCGCG CGCGCAATAAGCGCGCATTA TTAGCGATATATTATTAATCTAT A SEQ ID NO: 1378 GACTCGCATATAGGTATATCGCG CGCGCAATAAGCGCGCATTA TTAGCGATATATTATTAACCTAT A SEQ ID NO: 1379 GACTCGCATATAGGGATATCGCG CGCGCAATAAGCGCGCATT ATTAGCGATATATTATTACCCTA TA SEQ ID NO: 1380 GACTCGCATATAGGTATCACGCG CGCGCAATAAGCGCGCATT ATTAGCGTGATATTATTAACCTA TA SEQ ID NO: 1381 GACTCGCAGCCCGATATATAGCG CGCGCAATAAGCGCGCATT ATTAGCTATATATTATTAATCGG GC SEQ ID NO: 1382 GACTCGCAGCCCGATATATCGCG CGCGCAATAAGCGCGCATT ATTAGCGATATATTATTAATCGG GC SEQ ID NO: 1383 GACTCGCAGCCCGGTATATCGCG CGCGCAATAAGCGCGCATT ATTAGCGATATATTATTAACCGG GC SEQ ID NO: 1384 GACTCGCAGCCCGGGATATCGCG CGCGCAATAAGCGCGCATT ATTAGCGATATATTATTACCCGG GC SEQ ID NO: 1385 GACTCGCAGCCCGGTATCACGCG CGCGCAATAAGCGCGCATT ATTAGCGTGATATTATTAACCGG GC SEQ ID NO: 1386 GACTCGCATATAGATATATAGCG CGCGCAATAAGCGCGCATT ATTAGCTATATATTATTAATCTA TAATTATTATGCGAGT SEQ ID NO: 1387 GACTCGCATATAGATATATCGCG CGCGCAATAAGCGCGCATTA TTAGCGATATATTATTAATCTAT AATTATTATGCGAGT SEQ ID NO: 1388 GACTCGCATATAGGTATATCGCG CGCGCAATAAGCGCGCATTA TTAGCGATATATTATTAACCTAT AATTATTATGCGAGT SEQ ID NO: 1389 GACTCGCATATAGGGATATCGCG CGCGCAATAAGCGCGCATT ATTAGCGATATATTATTACCCTA TAATTATTATGCGAGT SEQ ID NO: 1390 GACTCGCATATAGGTATCACGCG CGCGCAATAAGCGCGCATT ATTAGCGTGATATTATTAACCTA TAATTATTATGCGAGT SEQ ID NO: 1391 GACTCGCAGCCCGATATATAGCG CGCGCAATAAGCGCGCATT ATTAGCTATATATTATTAATCGG GCATTATTATGCGAGT SEQ ID NO: 1392 GACTCGCAGCCCGATATATCGCG CGCGCAATAAGCGCGCATT ATTAGCGATATATTATTAATCGG GCATTATTATGCGAGT SEQ ID NO: 1393 GACTCGCAGCCCGGTATATCGCG CGCGCAATAAGCGCGCATT ATTAGCGATATATTATTAACCGG GCATTATTATGCGAGT SEQ ID NO: 1394 GACTCGCAGCCCGGGATATCGCG CGCGCAATAAGCGCGCATT ATTAGCGATATATTATTACCCGG GCATTATTATGCGAGT SEQ ID NO: 1395 GACTCGCAGCCCGGTATCACGCG CGCGCAATAAGCGCGCATT ATTAGCGTGATATTATTAACCGG GCATTATTATGCGAGT SEQ ID NO: 1396 GACTCGCAGATATGTATATAGCG CGCGCAATAAGCGCGCATT ATTAGCTATAATTATTATACATA SEQ ID NO: 1397 GACTCGCAGATATGTATATCGCG CGCGCAATAAGCGCGCATTA TTAGCGATAATTATTATACATA SEQ ID NO: 1398 GACTCGCAGATATGGATATCGCG CGCGCAATAAGCGCGCATT ATTAGCGATAATTATTATCCATA SEQ ID NO: 1399 GACTCGCAGATATGGATCACGCG CGCGCAATAAGCGCGCATT ATTAGCGTGAATTATTATCCATA SEQ ID NO: 1400 GACTCGCAGATATGGCACACGCG CGCGCAATAAGCGCGCATT ATTAGCGTGTATTATTAGCCATA SEQ ID NO: 1401 GACTCGCAGCCCGGTATATAGCG CGCGCAATAAGCGCGCATT ATTAGCTATAATTATTATACCGG SEQ ID NO: 1402 GACTCGCAGCCCGGTATATCGCG CGCGCAATAAGCGCGCATT ATTAGCGATAATTATTATACCGG SEQ ID NO: 1403 GACTCGCAGCCCGGGATATCGCG CGCGCAATAAGCGCGCATT ATTAGCGATAATTATTATCCCGG SEQ ID NO: 1404 GACTCGCAGCCCGGGATCACGCG CGCGCAATAAGCGCGCATT ATTAGCGTGAATTATTATCCCGG SEQ ID NO: 1405 GACTCGCAGCCCGGGCACACGCG CGCGCAATAAGCGCGCATT ATTAGCGTGTATTATTAGCCCGG SEQ ID NO: 1406 GACTCGCAGCCTGTGATATAGCG CGCGCAATAAGCGCGCATT ATTAGCTATATTATTAATCAC SEQ ID NO: 1407 GACTCGCAGCCTGTGATATCGCG CGCGCAATAAGCGCGCATTA TTAGCGATATTATTAATCAC SEQ ID NO: 1408 GACTCGCAGCCTGTGCATACGCG CGCGCAATAAGCGCGCATT ATTAGCGTAATTATTATGCAC SEQ ID NO: 1409 GACTCGCAGCCTGTGCACACGCG CGCGCAATAAGCGCGCATT ATTAGCGTGATTATTATGCAC SEQ ID NO: 1410 GACTCGCAGCCCGGGATATAGCG CGCGCAATAAGCGCGCATT ATTAGCTATATTATTAATCCC SEQ ID NO: 1411 GACTCGCAGCCCGGGATATCGCG CGCGCAATAAGCGCGCATT ATTAGCGATATTATTAATCCC SEQ ID NO: 1412 GACTCGCAGCCCGGGCATACGCG CGCGCAATAAGCGCGCATT ATTAGCGTAATTATTATGCCC SEQ ID NO: 1413 GACTCGCAGCCCGGGCACACGCG CGCGCAATAAGCGCGCATT ATTAGCGTGATTATTATGCCC SEQ ID NO: 1414 GACTCGCAGCCTGTGATATAGCG CGCGCAATAAGCGCGCATT ATTAGCTATATTATTAATCACAT TATTAAGGCTATTATTAGCG AG SEQ ID NO: 1415 GACTCGCAGCCTGTGATATCGCG CGCGCAATAAGCGCGCATTA TTAGCGATATTATTAATCACATT ATTAAGGCTATTATTAGCGA G SEQ ID NO: 1416 GACTCGCAGCCTGTGCATACGCG CGCGCAATAAGCGCGCATT ATTAGCGTAATTATTATGCACAT TATTAAGGCTATTATTAGCG AG SEQ ID NO: 1417 GACTCGCAGCCTGTGCACACGCG CGCGCAATAAGCGCGCATT ATTAGCGTGATTATTATGCACAT TATTAAGGCTATTATTAGCG AG SEQ ID NO: 1418 GACTCGCAGCCCGGGATATAGCG CGCGCAATAAGCGCGCATT ATTAGCTATATTATTAATCCCAT TATTAGGGCTATTATTAGCGA G SEQ ID NO: 1419 GACTCGCAGCCCGGGATATCGCG CGCGCAATAAGCGCGCATT ATTAGCGATATTATTAATCCCAT TATTAGGGCTATTATTAGCG AG SEQ ID NO: 1420 GACTCGCAGCCCGGGCATACGCG CGCGCAATAAGCGCGCATT ATTAGCGTAATTATTATGCCCAT TATTAGGGCTATTATTAGCG AG SEQ ID NO: 1421 GACTCGCAGCCCGGGCACACGCG CGCGCAATAAGCGCGCATT ATTAGCGTGATTATTATGCCCAT TATTAGGGCTATTATTAGCG AG *indicate the bonds that are phosphorothioate (PS) modified. These sequences may include nuclease resistant modifications such as PS modifications in all bases except the Loop sequences, where Loop sequences are the unhybridized bases. The number of modifications, e.g., PS, can vary from “0” to “max = total number of bases − number of bases in loops.

In any of the foregoing embodiments, the blocked nucleic acid molecules of the disclosure may further contain a reporter moiety attached thereto such that cleavage of the blocked nucleic acid releases a signal from the reporter moiety. (See FIG. 4 , mechanisms depicted at center and bottom.)

Also, in any of the foregoing embodiments, the blocked nucleic acid molecule may be a modified or non-naturally occurring nucleic acid molecule. In some embodiments, the blocked nucleic acid molecules of the disclosure may further contain a locked nucleic acid (LNA), a bridged nucleic acid (BNA), and/or a peptide nucleic acid (PNA). The blocked nucleic acid molecule may contain a modified or non-naturally occurring nucleoside, nucleotide, and/or internucleoside linkage, such as a 2′-O-methyl (2′—O-Me) modified nucleoside, a 2′-fluoro (2′-F) modified nucleoside, and a phosphorothioate (PS) bond, any other nucleic acid molecule modifications described above, and any combination thereof.

FIG. 2G at left shows an exemplary single-strand blocked nucleic acid molecule and how the configuration of this blocked nucleic acid molecule is able to block R-loop formation with an RNP complex, thereby blocking activation of the trans-cleavage activity of RNP2. The single-strand blocked nucleic acid molecule is self-hybridized and comprises: a target strand (TS) sequence complementary to the gRNA (e.g., crRNA) of RNP2; a cleavable non-target strand (NTS) sequence that is partially hybridized (e.g., it contains secondary loop structures) to the TS sequence; and a protospacer adjacent motif (PAM) sequence (e.g., 5′ NAAA 3′) that is specifically located at the 3′ end of the TS sequence. An RNP complex with 3′→+5′ diffusion (e.g., 1D diffusion) initiates R-loop formation upon PAM recognition. R-loop formation is completed upon a stabilizing ≥17 base hybridization of the TS to the gRNA of RNP2; however, because of the orientation of the PAM sequence relative to the secondary loop structure(s), the blocked nucleic acid molecule sterically prevents the TS sequence from hybridizing with the gRNA of RNP2, thereby blocking the stable R-loop formation required for the cascade reaction.

FIG. 2G at right shows the blocked nucleic acid molecule being unblocked via trans-cleavage (e.g., by RNP1) and subsequent dehybridization of the NTS's secondary loop structures, followed by binding of the TS sequence to the gRNA of RNP2, thereby completing stable R-loop formation and activating the trans-cleavage activity of the RNP2 complex.

In some embodiments, the blocked nucleic acid molecules provided herein are circular DNAs, RNAs or chimeric (DNA-RNA) molecules (FIG. 2H), and the blocked nucleic acid molecules may include different base compositions depending on the Cas enzyme used for RNP1 and RNP2. For the circular design of blocked nucleic acid molecules, the 5′ and 3′ ends are covalently linked together. This configuration makes internalization of the blocked nucleic acid molecule into RNP2—and subsequent RNP2 activation—sterically unfavorable, thereby blocking the progression of a CRISPR Cascade reaction. Thus, RNP2 activation (e.g., trans-cleavage activity) happens after cleavage of a portion of the blocked nucleic acid molecule followed by linearization and internalization of unblocked nucleic acid molecule into RNP2.

In some embodiments, the blocked nucleic acid molecules are topologically circular molecules with 5′ and 3′ portions hybridized to each other using DNA, RNA, LNA, BNA, or PNA bases which have a very high melting temperature (Tm). The high Tm causes the structure to effectively behave as a circular molecule even though the 5′ and 3′ ends are not covalently linked. The 5′ and 3′ ends can also have base non-naturally occurring modifications such as phosphorothioate bonds to provide increased stability.

In embodiments where the blocked nucleic acid molecules are circularized (e.g., circular or topologically circular), as illustrated in FIG. 2H, each blocked nucleic acid molecule includes a first region, which is a target sequence specific to the gRNA of RNP2, and a second region, which is a sequence that can be cleaved by nuclease enzymes of activated RNP1 and/or RNP2. The first region may include a nuclease-resistant nucleic acid sequence such as, for example, a phosphorothioate group or other non-naturally occurring nuclease-resistant base modifications, for protection from trans-endonuclease activity. In some embodiments, when the Cas enzyme in both RNP1 and RNP2 is Cas12a, the first region of the blocked nucleic acid molecule includes a nuclease-resistant DNA sequence, and the second region of the blocked nucleic acid molecule includes a cleavable DNA sequence. In other embodiments, when the Cas enzyme in RNP1 is Cas12a and the Cas enzyme in RNP2 is Cas13a, the first region of the blocked nucleic acid molecule includes a nuclease-resistant RNA sequence, and the second region of the blocked nucleic acid molecule includes a cleavable DNA sequence and a cleavable RNA sequence. In yet other embodiments, when the Cas enzyme in RNP1 is Cas13a and the Cas enzyme in RNP2 is Cas12a, the first region of the blocked nucleic acid molecule includes a nuclease-resistant DNA sequence, and the second region of the blocked nucleic acid molecule includes a cleavable DNA sequence and a cleavable RNA sequence. In some other embodiments, when the Cas enzyme in both RNP1 and RNP2 is Cas13a, the first region of the blocked nucleic acid molecule includes a nuclease-resistant RNA sequence, and the second region of the blocked nucleic acid molecule includes a cleavable RNA sequence.

The Cascade Assay Employing Blocked Primer Molecules

The blocked nucleic acids described above may also be blocked primer molecules. Blocked primer molecules include a sequence complementary to a primer binding domain (PBD) on a template molecule (see description below in reference to FIGS. 3A and 3B) and can have the same general structures as the blocked nucleic acid molecules described above. A PBD serves as a nucleotide sequence for primer hybridization followed by primer polymerization by a polymerase. In any of Formulas I, II, or III described above, the blocked primer nucleic acid molecule may include a sequence complementary to the PBD on the 5′ end of T. The unblocked primer nucleic acid molecule can bind to a template molecule at the PBD and copy the template molecule via polymerization by a polymerase.

Other specific embodiments of the cascade assay that utilize blocked primer molecules and are depicted in FIGS. 3A and 3B. In the embodiments using blocked nucleic acid molecules described above, activation of RNP1 and trans-cleavage of the blocked nucleic acid molecules were used to activate RNP2—that is, the unblocked nucleic acid molecules are a target sequence for the gRNA in RNP2. In contrast, in the embodiments using blocked primers, activation of RNP1 and trans-cleavage unblocks a blocked primer molecule that is then used to prime a template molecule for extension by a polymerase, thereby synthesizing activating molecules that are the target sequence for the gRNA in RNP2.

FIG. 3A is a diagram showing the sequence of steps in an exemplary cascade assay involving circular blocked primer molecules and linear template molecules. At left of FIG. 3A is a cascade assay reaction mix comprising 1) RNP1s (301) (only one RNP1 is shown); 2) RNP2s (302); 3) linear template molecules (330) (which is the non-target strand); 4) a circular blocked primer molecule (334) (i.e., a high K_(d) molecule); and 5) a polymerase (338), such as a D29 polymerase. The linear template molecule (330) (non-target strand) comprises a PAM sequence (331), a primer binding domain (PBD) (332) and, optionally, a nucleoside modification (333) to protect the linear template molecule (330) from 3′→5′ exonuclease activity. Blocked primer molecule (334) comprises a cleavable region (335) and a complement to the PBD (332) on the linear template molecule (330).

Upon addition of a sample comprising a target nucleic acid of interest (304) (capable of complexing with the gRNA in RNP1 (301)), the target nucleic acid of interest (304) combines with and activates RNP1 (305) but does not interact with or activate RNP2 (302). Once activated, RNP1 cuts the target nucleic acid of interest (304) via sequence specific cis-cleavage, which activates non-specific trans-cleavage of other nucleic acids present in the reaction mix, including at least one of the blocked primer molecules (334). The circular blocked primer molecule (334) (i.e., a high K_(d) molecule, where high K_(d) relates to binding to RNP2) upon cleavage becomes an unblocked linear primer molecule (344) (a low K_(d) molecule, where low K_(d) related to binding to RNP2), which has a region (336) complementary to the PBD (332) on the linear template molecule (330) and can bind to the linear template molecule (330).

Once the unblocked linear primer molecule (344) and the linear template molecule (330) are hybridized (i.e., hybridized at the PBD (332) of the linear template molecule (330) and the PBD complement (336) on the unblocked linear primer molecule (344)), 3′→5′ exonuclease activity of the polymerase (338) removes the unhybridized single-stranded DNA at the end of the unblocked primer molecule (344) and the polymerase (338) can copy the linear template molecule (330) to produce a synthesized activating molecule (346) (a complement of the non-target strand, which is a target strand). The synthesized activating molecule (346) is capable of activating RNP2 (302→308). As described above, because the nucleic acid-guided nuclease in the RNP2 (308) complex exhibits (that is, possesses) both cis- and trans-cleavage activity, more blocked primer molecules (334) become unblocked primer molecules (344) triggering activation of more RNP2s (308) and more trans-cleavage activity in a cascade. As stated above in relation to blocked and unblocked nucleic acid molecules (both linear and circular), the unblocked primer molecule has a higher binding affinity for the gRNA in RNP2 than does the blocked primer molecule, although there may be some “leakiness” where some blocked primer molecules are able to interact with the gRNA in RNP2. However, an unblocked primer molecule has a substantially higher likelihood than a blocked primer molecule to hybridize with the gRNA of RNP2.

FIG. 3A at bottom depicts the concurrent activation of reporter moieties. Intact reporter moieties (309) comprise a quencher (310) and a fluorophore (311). As described above in relation to FIG. 1B, the reporter moieties are also subject to trans-cleavage by activated RNP1 (305) and RNP2 (308). The intact reporter moieties (309) become activated reporter moieties (312) when the quencher (310) is separated from the fluorophore (311), and the fluorophore emits a fluorescent signal (313). Signal strength increases rapidly as more blocked primer molecules (334) become unblocked primer molecules (344) generating synthesized activating molecules (346) and triggering activation of more RNP2 (308) complexes and more trans-cleavage activity of the reporter moieties (309). Again, here the reporter moieties are shown as separate molecules from the blocked nucleic acid molecules, but other configurations may be employed and are discussed in relation to FIG. 4 . Also, as with the cascade assay embodiment utilizing blocked nucleic acid molecules that are not blocked primers, with the exception of the gRNA in RNP1, the cascade assay components stay the same no matter what target nucleic acid(s) of interest are being detected.

FIG. 3B is a diagram showing the sequence of steps in an exemplary cascade assay involving blocked primer molecules and circular template molecules. The cascade assay of FIG. 3B differs from that depicted in FIG. 3A by the configuration of the template molecule. Where the template molecule in FIG. 3A was linear, in FIG. 3B the template molecule is circular. At left of FIG. 3B is a cascade assay reaction mix comprising 1) RNP1s (301) (only one RNP1 is shown); 2) RNP2s (302); 3) a circular template molecule (352) (non-target strand); 4) a circular blocked primer molecule (334); and 5) a polymerase (338), such as a D29 polymerase. The circular template molecule (352) (non-target strand) comprises a PAM sequence (331) and a primer binding domain (PBD) (332). Blocked primer molecule (334) comprises a cleavable region (335) and a complement to the PBD (332) on the circular template molecule (352).

Upon addition of a sample comprising a target nucleic acid of interest (304) (capable of complexing with the gRNA in RNP1 (301)), the target nucleic acid of interest (304) combines with and activates RNP1 (305) but does not interact with or activate RNP2 (302). Once activated, RNP1 cuts the target nucleic acid of interest (304) via sequence specific cis-cleavage, which activates non-specific trans-cleavage of other nucleic acids present in the reaction mix, including at least one of the blocked primer molecules (334). The circular blocked primer molecule (334), upon cleavage, becomes an unblocked linear primer molecule (344), which has a region (336) complementary to the PBD (332) on the circular template molecule (352) and can hybridize with the circular template molecule (352).

Once the unblocked linear primer molecule (344) and the circular template molecule (352) are hybridized (i.e., hybridized at the PBD (332) of the circular template molecule (352) and the PBD complement (336) on the unblocked linear primer molecule (344)), 3′→5′ exonuclease activity of the polymerase (338) removes the unhybridized single-stranded DNA at the 3′ end of the unblocked primer molecule (344). The polymerase (338) can now use the circular template molecule (352) (non-target strand) to produce concatenated activating nucleic acid molecules (360) (which are concatenated target strands), which will be cleaved by the trans-cleavage activity of activated RNP1. The cleaved regions of the concatenated synthesized activating molecules (360) (target strand) are capable of activating the RNP2 (302→308) complex.

As described above, because the nucleic acid-guided nuclease in RNP2 (308) comprises both cis- and trans-cleavage activity, more blocked primer molecules (334) become unblocked primer molecules (344) triggering activation of more RNP2s (308) and more trans-cleavage activity in a cascade. FIG. 3B at bottom depicts the concurrent activation of reporter moieties. Intact reporter moieties (309) comprise a quencher (310) and a fluorophore (311). As described above in relation to FIG. 1B, the reporter moieties are also subject to trans-cleavage by activated RNP1 (305) and RNP2 (308). The intact reporter moieties (309) become activated reporter moieties (312) when the quencher (310) is separated from the fluorophore (311), and the fluorescent signal (313) is unquenched and can be detected. Signal strength increases rapidly as more blocked primer molecules (334) become unblocked primer molecules (344) generating synthesized activating nucleic acid molecules and triggering activation of more RNP2s (308) and more trans-cleavage activity of the reporter moieties (309). Again, here the reporter moieties are shown as separate molecules from the blocked nucleic acid molecules, but other configurations may be employed and are discussed in relation to FIG. 4 . Also note that as with the other embodiments of the cascade assay, in this embodiment, with the exception of the gRNA in RNP1, the cascade assay components stay the same no matter what target nucleic acid(s) of interest are being detected.

The polymerases used in the “blocked primer molecule” embodiments serve to polymerize a reverse complement strand of the template molecule (non-target strand) to generate a synthesized activating molecule (target strand) as described above. In some embodiments, the polymerase is a DNA polymerase, such as a BST, T4, or Therminator polymerase (New England BioLabs Inc., Ipswich Mass., USA). In some embodiments, the polymerase is a Klenow fragment of a DNA polymerase. In some embodiments the polymerase is a DNA polymerase with 5′→3′ DNA polymerase activity and 3′→5′ exonuclease activity, such as a Type I, Type II, or Type III DNA polymerase. In some embodiments, the DNA polymerase, including the Phi29, T7, Q5®, Q5U®, Phusion®, OneTaq®, LongAmp®, Vent®, or Deep Vent® DNA polymerases (New England BioLabs Inc., Ipswich Mass., USA), or any active portion or variant thereof. Also, a 3′ to 5′ exonuclease can be separately used if the polymerase lacks this activity.

FIG. 4 depicts three mechanisms in which a cascade assay reaction can release a signal from a reporter moiety. FIG. 4 at top shows the mechanism discussed in relation to FIGS. 2A, 3A and 3B. In this embodiment, a reporter moiety 409 is a separate molecule from the blocked nucleic acid molecules present in the reaction mix. Reporter moiety (409) comprises a quencher (410) and a fluorophore (411). An activated reporter moiety (412) emits a signal from the fluorophore (411) once it has been physically separated from the quencher (410).

FIG. 4 at center shows a blocked nucleic acid molecule (403), which is also a reporter moiety. In addition to quencher (410) and fluorophore (411), a blocking moiety (407) can be seen (see also blocked nucleic acid molecules 203 in FIG. 2A). Blocked nucleic acid molecule/reporter moiety (403) comprises a quencher (410) and a fluorophore (411). In this embodiment of the cascade assay, when the blocked nucleic acid molecule (403) is unblocked due to trans-cleavage initiated by the target nucleic acid of interest binding to RNP1, the unblocked nucleic acid molecule (406) also becomes an activated reporter moiety with fluorophore (411) separated from quencher (410). Note both the blocking moiety (407) and the quencher (410) are removed. In this embodiment, reporter signal is directly generated as the blocked nucleic acid molecules become unblocked.

FIG. 4 at the bottom shows that cis-cleavage of an unblocked nucleic acid or a synthesized activation molecule at a PAM distal sequence by RNP2 generates a signal. Shown are activated RNP2 (408), unblocked nucleic acid molecule (461), quencher (410), and fluorophore (411) forming an activated RNP2 with the unblocked nucleic acid/reporter moiety intact (460). Cis-cleavage of the unblocked nucleic acid/reporter moiety (461) results in an activated RNP2 with the reporter moiety activated (462), comprising the activated RNP2 (408), the unblocked nucleic acid molecule with the reporter moiety activated (463), quencher (410) and fluorophore (411).

Applications of the Cascade Assay

The present disclosure describes cascade assays for detecting a target nucleic acid of interest in a sample. As described above, the various embodiments of the cascade assay are notable in that, with the exception of the gRNA in RNP1, the cascade assay components stay the same no matter what target nucleic acid(s) of interest are being detected.

Target nucleic acids of interest are derived from samples. Suitable samples for testing include, but are not limited to, any environmental sample, such as air, water, soil, surface, food, clinical sites and products, industrial sites and products, pharmaceuticals, medical devices, nutraceuticals, cosmetics, personal care products, agricultural equipment and sites, and commercial samples, and any biological sample obtained from an organism or a part thereof, such as a plant, animal, or bacteria. In some embodiments, the biological sample is obtained from an animal subject, such as a human subject. A biological sample is any solid or fluid sample obtained from, excreted by or secreted by any living organism, including, without limitation, single celled organisms, such as bacteria, yeast, protozoans, and amoebas among others, multicellular organisms including plants or animals, including samples from a healthy or apparently healthy human subject or a human patient affected by a condition or disease to be diagnosed or investigated, such as an infection with a pathogenic microorganism, such as a pathogenic bacteria or virus. For example, a biological sample can be a biological fluid obtained from, for example, blood, plasma, serum, urine, stool, sputum, mucous, lymph fluid, synovial fluid, bile, ascites, pleural effusion, seroma, saliva, cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion, a transudate, an exudate (for example, fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint (for example, a normal joint or a joint affected by disease, such as rheumatoid arthritis, osteoarthritis, gout or septic arthritis), or a swab of skin or mucosal membrane surface (e.g., a nasal or buccal swab).

In some embodiments, the sample can be a viral or bacterial sample or a biological sample that has been minimally processed, e.g., only treated with a brief lysis step prior to detection. In some embodiments, minimal processing can include thermal lysis at an elevated temperature to release nucleic acids. Suitable methods are contemplated in U.S. Pat. No. 9,493,736, among other references. Common methods for cell lysis involve thermal, chemical, enzymatic, or mechanical treatment of the sample or a combination of those. In some embodiments, minimal processing can include treating the sample with chaotropic salts such as guanidine isothiocyanate or guanidine HCl. Suitable methods are contemplated in U.S. Pat. Nos. 8,809,519, 7,893,251, among other references. In some embodiments, minimal processing may include contacting the sample with reducing agents such as DTT or TCEP and EDTA to inactivate inhibitors and/or other nucleases present in the crude samples. In other embodiments, minimal processing for biofluids may include centrifuging the samples to obtain cell-debris free supernatant before applying the reagents. Suitable methods are contemplated in U.S. Pat. No. 8,809,519, among other references. In still other embodiments, minimal processing may include performing DNA/RNA extraction to get purified nucleic acids before applying CRISPR Cascade reagents.

FIG. 5A shows a lateral flow assay (LFA) device that can be used to detect the cleavage and separation of a signal from a reporter moiety. For example, the reporter moiety may be a single-stranded or double-stranded oligonucleotide with terminal biotin and fluorescein amidite (FAM) modifications; and, as described above, the reporter moiety may also be part of a blocked nucleic acid. The LFA device may include a pad with binding particles, such as gold nanoparticles functionalized with anti-FAM antibodies; a control line with a first binding moiety attached, such as avidin or streptavidin; a test line with a second binding moiety attached, such as antibodies; and an absorption pad. After completion of a cascade assay (see FIGS. 2A, 3A, and 3B), the assay reaction mix is added to the pad containing the binding particles, (e.g., antibody labeled gold nanoparticles). When the target nucleic acid of interest is present, a reporter moiety is cleaved, and when the target nucleic acid of interest is absent, the reporter is not cleaved.

A moiety on the reporter binds to the binding particles and is transported to the control line. When the target nucleic acid of interest is absent, the reporter moiety is not cleaved, and the first binding moiety binds to the reporter moiety, with the binding particles attached. When the target nucleic acid of interest is present, one portion of the cleaved reporter moiety binds to the first binding moiety, and another portion of the cleaved reporter moiety bound to the binding particles via the moiety binds to the second binding moiety. In one example, anti-FAM gold nanoparticles bind to a FAM terminus of a reporter moiety and flow sequentially towards the control line and then to the test line. For reporters that are not trans-cleaved, gold nanoparticles attach to the control line via biotin-streptavidin and result in a dark control line. In a negative test, since the reporter has not been cleaved, all gold conjugates are trapped on control line due to attachment via biotin-streptavidin. A negative test will result in a dark control line with a blank test line. In a positive test, reporter moieties have been trans-cleaved by the cascade assay, thereby separating the biotin terminus from the FAM terminus. For cleaved reporter moieties, nanoparticles are captured at the test line due to anti-FAM antibodies. This positive test results in a dark test line in addition to a dark control line.

In some embodiments, the LFA device is designed for syndromic testing. For example, multiple strips with pooled RNP1s targeting different target nucleic acids of interest may be employed, either as separate devices or in a combined device. As a non-limiting example, a syndromic testing device could include four lateral flow strips, with each strip indicating the presence of at least one out of several generally related (e.g., by genetics or by treatment) pathogens (FIG. 5B). One example of a use for syndromic testing is in respiratory illness. For example, the first lateral flow strip could indicate the presence of at least one of the several strains of influenza that cause the common flu (e.g., influenza A, influenza A/H1, influenza A/H3, influenza A/H1-2009, and influenza B); the second lateral flow strip could indicate the presence of at least one of the multiple strains of respiratory syncytial virus (RSV), such as RSV-A and RSV-B; the third lateral flow strip could indicate the presence of at least one variant of SARS-CoV-2 (e.g., B.1.1.7, B.1.351, P.1, B.1.617.2, BA.1, BA.2, BA.2.12.1, BA.4, and BA.5); and the fourth lateral flow strip could indicate the presence of at least one of other pathogens of interest (e.g., parainfluenza virus 1-4, human metapneumovirus, human rhinovirus, human enterovirus, adenovirus, coronavirus HKU1, coronavirus NL63, coronavirus 229E, coronavirus OC43, MERS, and many more). The results shown in FIG. 5B indicate a positive test for the presence of RSVA and/or RSV B nucleic acid molecules. Also as seen in FIG. 5B, the syndromic testing device could further include a lateral flow strip for a negative control and a lateral flow strip for a positive control.

The components of the cascade assay may be provided in various kits. In one aspect, the kit for detecting a target nucleic acid of interest in a sample includes: first ribonucleoprotein complexes (RNP1s), second ribonucleoprotein complexes (RNP2s), blocked nucleic acid molecules, and reporter moieties. The first complex (RNP1) comprises a first nucleic acid-guided nuclease and a first gRNA, where the first gRNA includes a sequence complementary to the target nucleic acid(s) of interest. Binding of the first complex (RNP1) to the target nucleic acid(s) of interest activates trans-cleavage activity of the first nucleic acid-guided nuclease. The second complex (RNP2) comprises a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest. The blocked nucleic acid molecule comprises a sequence complementary to the second gRNA, where trans-cleavage of the blocked nucleic acid molecule results in an unblocked nucleic acid molecule and the unblocked nucleic acid molecule can bind to the second complex (RNP2), thereby activating the trans-cleavage activity of the second nucleic acid-guided nuclease. Activating trans-cleavage activity in RNP2 results in an exponential increase in unblocked nucleic acid molecules and in active reporter moieties, where reporter moieties are nucleic acid molecules and/or are operably linked to the blocked nucleic acid molecules and produce a detectable signal upon cleavage by RNP2.

In a second aspect, the kit for detecting a target nucleic acid molecule in sample includes: first ribonucleoprotein complexes (RNP1s), second ribonucleoprotein complexes (RNP2s), template molecules, blocked primer molecules, a polymerase, NTPs, and reporter moieties. The first ribonucleoprotein complex (RNP1) comprises a first nucleic acid-guided nuclease and a first gRNA, where the first gRNA includes a sequence complementary to the target nucleic acid of interest and where binding of RNP1 to the target nucleic acid(s) of interest activates trans-cleavage activity of the first nucleic acid-guided nuclease. The second complex (RNP2) comprises a second nucleic acid-guided nuclease and a second gRNA that is not complementary to the target nucleic acid of interest. The template molecules comprise a primer binding domain (PBD) sequence as well as a sequence corresponding to a spacer sequence of the second gRNA. The blocked primer molecules comprise a sequence that is complementary to the PBD on the template nucleic acid molecule and a blocking moiety.

Upon binding to the target nucleic acid of interest, RNP1 becomes active triggering trans-cleavage activity that cuts at least one of the blocked primer molecules to produce at least one unblocked primer molecule. The unblocked primer molecule hybridizes to the PBD of one of the template nucleic acid molecules, is trimmed of excess nucleotides by the 3′-to-5′ exonuclease activity of the polymerase and is then extended by the polymerase and NTPs to form a synthesized activating molecule with a sequence that is complementary to the second gRNA of RNP2. Upon activating RNP2, additional trans-cleavage activity is initiated, cleaving at least one additional blocked primer molecule. Continued cleavage of blocked primer molecules and subsequent activation of more RNP2s proceeds at an exponential rate. A signal is generated upon cleavage of a reporter molecule by active RNP2 complexes; therefore, a change in signal production indicates the presence of the target nucleic acid molecule.

Any of the kits described herein may further include a sample collection device, e.g., a syringe, lancet, nasal swab, or buccal swab for collecting a biological sample from a subject, and/or a sample preparation reagent, e.g., a lysis reagent. Each component of the kit may be in separate container or two or more components may be in the same container. The kit may further include a lateral flow device used for contacting the biological sample with the reaction mixture, where a signal is generated to indicate the presence or absence of the target nucleic acid molecule of interest. In addition, the kit may further include instructions for use and other information.

EXAMPLES

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent or imply that the experiments below are all of or the only experiments performed. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific aspects without departing from the spirit or scope of the invention as broadly described. The present aspects are, therefore, to be considered in all respects as illustrative and not restrictive.

Example I: Preparation of Nucleic Acids of Interest

Mechanical lysis: Nucleic acids of interest may be isolated by various methods depending on the cell type and source (e.g., tissue, blood, saliva, environmental sample, etc.). Mechanical lysis is a widely-used cell lysis method and may be used to extract nucleic acids from bacterial, yeast, plant and mammalian cells. Cells are disrupted by agitating a cell suspension with “beads” at high speeds (beads for disrupting various types of cells can be sourced from, e.g., OPS Diagnostics (Lebanon N.J., US) and MP Biomedicals (Irvine, Calif., USA)). Mechanical lysis via beads begins with harvesting cells in a tissue or liquid, where the cells are first centrifuged and pelleted. The supernatant is removed and replaced with a buffer containing detergents as well as lysozyme and protease. The cell suspension is mixed to promote breakdown of the proteins in the cells and the cell suspension then is combined with small beads (e.g., glass, steel, or ceramic beads) that are mixed (e.g., vortexed) with the cell suspension at high speeds. The beads collide with the cells, breaking open the cell membrane with shear forces. After “bead beating”, the cell suspension is centrifuged to pellet the cellular debris and beads, and the supernatant may be purified via a nucleic acid binding column (such as the MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit from ThermoFisher (Waltham, Mass., USA) and others from Qiagen (Hilden Germany), TakaraBio (San Jose, Calif., USA), and Biocomma (Shenzen, China)) to collect the nucleic acids (see the discussion of solid phase extraction below).

Solid phase extraction (SPE): Another method for capturing nucleic acids is through solid phase extraction. SPE involves a liquid and stationary phase, which selectively separate the target analyte (here, nucleic acids) from the liquid in which the cells are suspended based on specific hydrophobic, polar, and/or ionic properties of the target analyte in the liquid and the stationary solid matrix. Silica binding columns and their derivatives are the most commonly used SPE techniques, having a high binding affinity for DNA under alkaline conditions and increased salt concentration; thus, a highly alkaline and concentrated salt buffer is used. The nucleic acid sample is centrifuged through a column with a highly porous and high surface area silica matrix, where binding occurs via the affinity between negatively charged nucleic acids and positively charged silica material. The nucleic acids bind to the silica matrices, while the other cell components and chemicals pass through the matrix without binding. One or more wash steps typically are performed after the initial sample binding (i.e., the nucleic acids to the matrix), to further purify the bound nucleic acids, removing excess chemicals and cellular components non-specifically bound to the silica matrix. Alternative versions of SPE include reverse SPE and ion exchange SPE, and use of glass particles, cellulose matrices, and magnetic beads.

Thermal lysis: Thermal lysis involves heating a sample of mammalian cells, virions, or bacterial cells at high temperatures thereby damaging the cellular membranes by denaturizing the membrane proteins. Denaturizing the membrane proteins results in the release of intracellular DNA. Cells are generally heated above 90° C., however time and temperature may vary depending on sample volume and sample type. Once lysed, typically one or more downstream methods, such as use of nucleic acid binding columns for solid phase extraction as described above, are required to further purify the nucleic acids.

Physical lysis: Common physical lysis methods include sonication and osmotic shock. Sonication involves creating and rupturing of cavities or bubbles to release shockwaves, thereby disintegrating the cellular membranes of the cells. In the sonication process, cells are added into lysis buffer, often containing phenylmethylsulfonyl fluoride, to inhibit proteases. The cell samples are then placed in a water bath and a sonication wand is placed directly into the sample solution. Sonication typically occurs between 20-50 kHz, causing cavities to be formed throughout the solution as a result of the ultrasonic vibrations; subsequent reduction of pressure then causes the collapse of the cavity or bubble resulting in a large amount of mechanical energy being released in the form of a shockwave that propagates through the solution and disintegrates the cellular membrane. The duration of the sonication pulses and number of pulses performed varies depending on cell type and the downstream application. After sonication, the cell suspension typically is centrifuged to pellet the cellular debris and the supernatant containing the nucleic acids may be further purified by solid phase extraction as described above.

Another form of physical lysis is osmotic shock, which is most typically used with mammalian cells. Osmotic shock involves placing cells in DI/distilled water with no salt added. Because the salt concentration is lower in the solution than in the cells, water is forced into the cell causing the cell to burst, thereby rupturing the cellular membrane. The sample is typically purified and extracted by techniques such as e.g., solid phase extraction or other techniques known to those of skill in the art.

Chemical lysis: Chemical lysis involves rupturing cellular and nuclear membranes by disrupting the hydrophobic-hydrophilic interactions in the membrane bilayers via detergents. Salts and buffers (such as, e.g., Tris-HCl pH8) are used to stabilize pH during extraction, and chelating agents (such as ethylenediaminetetraacetic acid (EDTA)) and inhibitors (e.g., Proteinase K) are also added to preserve the integrity of the nucleic acids and protect against degradation. Often, chemical lysis is used with enzymatic disruption methods (see below) for lysing bacterial cell walls. In addition, detergents are used to lyse and break down cellular membranes by solubilizing the lipids and membrane proteins on the surface of cells. The contents of the cells include, in addition to the desired nucleic acids, inner cellular proteins and cellular debris. Enzymes and other inhibitors are added after lysis to inactivate nucleases that may degrade the nucleic acids. Proteinase K is commonly added after lysis, destroying DNase and RNase enzymes capable of degrading the nucleic acids. After treatment with enzymes, the sample is centrifuged, pelleting cellular debris, while the nucleic acids remain in the solution. The nucleic acids may be further purified as described above.

Another form of chemical lysis is the widely-used procedure of phenol-chloroform extraction. Phenol-chloroform extraction involves the ability for nucleic acids to remain soluble in an aqueous solution in an acidic environment, while the proteins and cellular debris can be pelleted down via centrifugation. Phenol and chloroform ensure a clear separation of the aqueous and organic (debris) phases. For DNA, a pH of 7-8 is used, and for RNA, a more acidic pH of 4.5 is used.

Enzymatic lysis: Enzymatic disruption methods are commonly combined with other lysis methods such as those described above to disrupt cellular walls (bacteria and plants) and membranes. Enzymes such as lysozyme, lysostaphin, zymolase, and protease are often used in combination with other techniques such as physical and chemical lysis. For example, one can use cellulase to disrupt plant cell walls, lysosomes to disrupt bacterial cell walls and zymolase to disrupt yeast cell walls.

Example II: RNP Formation

For RNP complex formation, 250 nM of LbCas12a nuclease protein was incubated with 375 nM of a target specific gRNA in 1× Buffer (10 mM Tris-HCl, 100 μg/mL BSA) with 2-15 mM MgCl₂ at 25° C. for 20 minutes. The total reaction volume was 2 μL. Other ratios of LbCas12a nuclease to gRNAs were tested, including 1:1, 1:2 and 1:5. The incubation temperature can range from 20° C.-37° C., and the incubation time can range from 10 minutes to 4 hours.

Example III: Blocked Nucleic Acid Molecule Formation

Ramp cooling: For formation of the secondary structure of blocked nucleic acids, 2.5 μM of a blocked nucleic acid molecule (any of Formulas I-IV) was mixed in a T50 buffer (20 mM Tris HCl, 50 mM NaCl) with 10 mM MgCl₂ for a total volume of 50 μL. The reaction was heated to 95° C. at 1.6° C./second and incubated at 95° C. for 5 minutes to dehybridize any secondary structures. Thereafter, the reaction was cooled to 37° C. at 0.015° C./second to form the desired secondary structure.

Snap cooling: For formation of the secondary structure of blocked nucleic acids, 2.5 μM of a blocked nucleic acid molecule (any of Formulas I-IV) was mixed in a T50 buffer (20 mM Tris HCl, 50 mM NaCl) with 10 mM MgCl₂ for a total volume of 50 μL. The reaction was heated to 95° C. at 1.6° C./second and incubated at 95° C. for 5 minutes to dehybridize any secondary structures. Thereafter, the reaction was cooled to room temperature by removing the heat source to form the desired secondary structure.

Snap cooling on ice: For formation of the secondary structure of blocked nucleic acids, 2.5 μM of a blocked nucleic acid molecule (any of Formulas I-IV) was mixed in a T50 buffer (20 mM Tris HCl, 50 mM NaCl) with 10 mM MgCl₂ for a total volume of 50 μL. The reaction was heated to 95° C. at 1.6° C./second and incubated at 95° C. for 5 minutes to dehybridize any secondary structures. Thereafter, the reaction was cooled to room temperature by placing the reaction tube on ice to form the desired secondary structure.

Example IV: Reporter Moiety Formation

The reporter moieties used in the reactions herein were single-stranded DNA oligonucleotides 5-10 bases in length (e.g., with sequences of TTATT, TTTATTT, ATTAT, ATTTATTTA, AAAAA, or AAAAAAAAA) with a fluorophore and a quencher attached on the 5′ and 3′ ends, respectively. In one example using a Cas12a cascade, the fluorophore was FAM-6, and the quencher was IOWA BLACK® (Integrated DNA Technologies, Coralville, Iowa). In another example using a Cas13 cascade, the reporter moieties were single stranded RNA oligonucleotides 5-10 bases in length (e.g., r(U)n, r(UUAUU)n, r(A)n).

Example V: Cascade Assay

9+1 Format (final reaction mix components added at the same time): RNP1 was assembled using the LbCas12a nuclease and a gRNA for the Methicillin resistant Staphylococcus aureus (MRSA) DNA according to the RNP complex formation protocol described in Example II (for this sequence, see Example VIII). Briefly, 250 nM LbCas12a nuclease was assembled with 375 nM of the MRSA-target specific gRNA. Next, RNP2 was formed using the LbCas12a nuclease and a gRNA specific for a selected blocked nucleic acid molecule (Formula I-IV) using 500 nM LbCas12a nuclease assembled with 750 nM of the blocked nucleic acid-specific gRNA incubated in 1×NEB 2.1 Buffer (New England Biolabs, Ipswich, Mass.) with 5 mM MgCl₂ at 25° C. for 20-40 minutes. Following incubation, RNP1s were diluted to a concentration of 75 nM LbCas12a: 112.5 nM gRNA. Thereafter, the final reaction was carried out in 1× Buffer, with 500 nM of the ssDNA reporter moiety, 1×ROX dye (Thermo Fisher Scientific, Waltham, Mass.) for passive reference, 2.5 mM MgCl₂, 4 mM NaCl, 15 nM LbCas12a: 22.5 nM gRNA RNP1, 20 nM LbCas12a: 35 nM gRNA RNP2, and 50 nM blocked nucleic acid molecule (any one of Formula I-IV) in a total volume of 9 μL. 1 μL of MRSA DNA target (with samples having as low as three copies and as many as 30000 copies—see FIGS. 6-14 ) was added to make a final volume of 10 μL. The final reaction was incubated in a thermocycler at 25° C. with fluorescence measurements taken every 1 minute.

2+1+7 Format (RNP1 and MRSA target pre-incubated before addition to final reaction mix): RNP1 was assembled using the LbCas12a nuclease and a gRNA for the MRSA DNA according to RNP formation protocol described in Example II (for this sequence, see Example VIII). Briefly, 250 nM LbCas12a nuclease was assembled with 375 nM of the MRSA-target specific gRNA. Next, RNP2 was formed using the LbCas12a nuclease and a gRNA specific for a selected blocked nucleic acid molecule (Formula I-IV) using 500 nM LbCas12a nuclease assembled with 750 nM of the blocked nucleic acid-specific gRNA incubated in 1×NEB 2.1 Buffer (New England Biolabs, Ipswich, Mass.) with 5 mM MgCl₂ at 25° C. for 20-40 minutes. Following incubation, RNP1s were diluted to a concentration of 75 nM LbCas12a: 112.5 nM gRNA. After dilution, the formed RNP1 was mixed with 1 μL of MRSA DNA target and incubated at 20° C.-37° C. for up to 10 minutes to activate RNP1. The final reaction was carried out in 1× Buffer, with 500 nM of the ssDNA reporter moiety, 1×ROX dye (Thermo Fisher Scientific, Waltham, Mass.) for passive reference, 2.5 mM MgCl₂, 4 mM NaCl, the pre-incubated and activated RNP1, 20 nM LbCas12a: 35 nM gRNA RNP2, and 50 nM blocked nucleic acid molecule (any one of Formula I-IV) in a total volume of 9 μL. The final reaction was incubated in a thermocycler at 25° C. with fluorescence measurements taken every 1 minute.

2+1+6+1 Format (RNP1 and MRSA target pre-incubated before addition to final reaction mix and blocked nucleic acid molecule added to final reaction mix last): RNP1 was assembled using the LbCas12a nuclease and a gRNA for the MRSA DNA according to the RNP complex formation protocol described in Example II (for this sequence, see Example VIII). Briefly, 250 nM LbCas12a nuclease was assembled with 375 nM of the MRSA-target specific gRNA. Next, RNP2 was formed using the LbCas12a nuclease and a gRNA specific for a selected blocked nucleic acid molecule (Formula I-IV) using 500 nM LbCas12a nuclease assembled with 750 nM of the blocked nucleic acid-specific gRNA incubated in 1×NEB 2.1 Buffer (New England Biolabs, Ipswich, Mass.) with 5 mM MgCl₂ at 25° C. for 20-40 minutes. Following incubation, RNP1s were diluted to a concentration of 75 nM LbCas12a: 112.5 nM gRNA. After dilution, the formed RNP1 was mixed with 1 μL of MRSA DNA target and incubated at 20° C.-37° C. for up to 10 minutes to activate RNP1. The final reaction was carried out in 1× Buffer, with 500 nM of the ssDNA reporter moiety, 1×ROX dye (Thermo Fisher Scientific, Waltham, Mass.) for passive reference, 2.5 mM MgCl₂, 4 mM NaCl, the pre-incubated and activated RNP1, and 20 nM LbCas12a: 35 nM gRNA RNP2 in a total volume of 9 μL. Once the reaction mix was made, 1 μL (50 nM) blocked nucleic acid molecule (any one of Formula I-IV) was added for a total volume of 10 μL. The final reaction was incubated in a thermocycler at 25° C. with fluorescence measurements taken every 1 minute.

Example VI: Detection of SARS-CoV-2 with the Cascade Assay in Under 10 Minutes

To detect the presence of SARS-CoV-2 in a sample and determine the sensitivity of detection with the cascade assay, titration experiments were performed using a SARS-CoV-2 gamma-inactivated virus and a synthesized positive control. To serve as the positive control for the detection system, a plasmid containing a 316 bp SARS-CoV-2 nucleocapsid gene (N-gene) was synthesized by IDT (Integrated DNA Technologies, Coralville, Iowa). The N-gene sequence was as follows.

SARS-CoV-2 N-gene Target Sequence (Positive Control; SEQ ID NO: 3): CTCAAGGAACAACATTGCCAAAAGGCTTCTACGCAGAAGGGAGCAGAGG CGGCAGTCAAGCCTCTTCTCGTTCCTCATCACGTAGTCGCAACAGTTCA AGAAATTCAACTCCAGGCAGCAGTAGGGGAACTTCTCCTGCTAGAATGG CTGGCAATGGCGGTGATGCTGCTCTTGCTTTGCTGCTGCTTGACAGATT GAACCAGCTTGAGAGCAAAATGTCTGGTAAAGGCCAACAACAACAAGGC CAAACTGTCACTAAGAAATCTGCTGCTGAGGCTTCTAAGAAGCCTCGGC AAAAACGTACTGCCACTAAAGC

For the detection of SARS-CoV-2, a gamma-inactivated virus was incubated in a buffer at 95° C. for 1 minute in order to lyse and release viral RNA, followed by reverse transcription to convert the viral RNA to cDNA. The reverse transcription primer is designed to reverse transcribe the SARS-CoV-2 N-gene. The reverse transcription primer is as follows.

Reverse Transcription Primer (SEQ ID NO: 4): GTTTGGCCTTGTTGTTGTT RNP1 was preassembled with a guide RNA (gRNA) sequence designed to target the N-gene of SARS-CoV-2. The guide sequence is as follows.

Guide Sequence (SEQ ID NO: 5): UAAUUUCUACUAAGUGUAGAUUUGAACUGUUGCGACUACGUGAU

RNP2 was preassembled with a gRNA sequence designed to target an unblocked nucleic acid molecule that results from unblocking (i.e., linearlizing) a circularized blocked nucleic acid molecule. A circularized blocked nucleic acid molecule was designed and synthesized. The blocked nucleic acid molecule was as follows.

Blocked nucleic acid molecule (SEQ ID NO: 6): GTT*AT*TA*AA*TG*AC*TT*CT*CATT where the * indicate bonds that are phosphorothioate modified. The 5′ and 3′ ends were covalently linked to form a circularized molecule. The SARS-CoV-2 gamma-inactivated virus or positive control with 1700, 170, 17, or 5 total copies of N-gene DNA, or a negative control (0 copies of N-gene), were added to a reaction mixture to begin the cascade assay. The reaction mix contained the preassembled RNP1, preassembled RNP2, a blocked nucleic acid molecule in a buffer (˜pH 8) containing 4 mM MgCl₂ and 101 mM NaCl. The buffering conditions were optimized to reduce non-specific nickase activity by the RNP complexes.

The cascade assay reaction proceeded for 20 minutes at 37° C. and fluorescence from the reporter molecule was measured. In all the SARS-CoV-2 gamma-inactivated virus and positive control titrations, a significant change in fluorescence was observed after 10 and 5 minutes, relative to the negative control (see the results in FIGS. 6 and 7 ). For the results shown in FIG. 6 , the presence of the N-gene was detected in 10 minutes or less at 37° C. The data represent 3 independent biological replicates. Data is presented as mean±s.d.****=p<0.0001 (student t-test). For the results shown in FIG. 7 , the presence of SARS-CoV-2 was detected in 10 minutes or 5 minutes at 37° C. The data represent 3 independent biological replicates. Data is presented as mean±s.d.****=p<0.0001 (student t-test). The results indicate that the cascade assay can detect as few as 5 SARS-CoV-2 target molecules in 10 minutes or less at room temperature.

Example VII: Detection of MRSA in 5 Minutes with Cascade Assay at 37° C.

To detect the presence of Methicillin resistant Staphylococcus aureus (MRSA) and determine the sensitivity of detection with the cascade assay, titration experiments with a MRSA DNA target nucleic acid of interest were performed. The MRSA DNA sequence (NCBI Reference Sequence NC: 007793.1) is as follows.

SEQ ID NO: 7: ATGAAAAAGATAAAAATTGTTCCACTTATTTTAATAGTTGTAGTTGTCGGGTTTGGTATATATTTTTATG CTTCAAAAGATAAAGAAATTAATAATACTATTGATGCAATTGAAGATAAAAATTTCAAACAAGTTTATAA AGATAGCAGTTATATTTCTAAAAGCGATAATGGTGAAGTAGAAATGACTGAACGTCCGATAAAAATATAT AATAGTTTAGGCGTTAAAGATATAAACATTCAGGATCGTAAAATAAAAAAAGTATCTAAAAATAAAAAAC GAGTAGATGCTCAATATAAAATTAAAACAAACTACGGTAACATTGATCGCAACGTTCAATTTAATTTTGT TAAAGAAGATGGTATGTGGAAGTTAGATTGGGATCATAGCGTCATTATTCCAGGAATGCAGAAAGACCAA AGCATACATATTGAAAATTTAAAATCAGAACGTGGTAAAATTTTAGACCGAAACAATGTGGAATTGGCCA ATACAGGAACAGCATATGAGATAGGCATCGTTCCAAAGAATGTATCTAAAAAAGATTATAAAGCAATCGC TAAAGAACTAAGTATTTCTGAAGACTATATCAAACAACAAATGGATCAAAATTGGGTACAAGATGATACC TTCGTTCCACTTAAAACCGTTAAAAAAATGGATGAATATTTAAGTGATTTCGCAAAAAAATTTCATCTTA CAACTAATGAAACAGAAAGTCGTAACTATCCTCTAGGAAAAGCGACTTCACATCTATTAGGTTATGTTGG TCCCATTAACTCTGAAGAATTAAAACAAAAAGAATATAAAGGCTATAAAGATGATGCAGTTATTGGTAAA AAGGGACTCGAAAAACTTTACGATAAAAAGCTCCAACATGAAGATGGCTATCGTGTCACAATCGTTGACG ATAATAGCAATACAATCGCACATACATTAATAGAGAAAAAGAAAAAAGATGGCAAAGATATTCAACTAAC TATTGATGCTAAAGTTCAAAAGAGTATTTATAACAACATGAAAAATGATTATGGCTCAGGTACTGCTATC CACCCTCAAACAGGTGAATTATTAGCACTTGTAAGCACACCTTCATATGACGTCTATCCATTTATGTATG GCATGAGTAACGAAGAATATAATAAATTAACCGAAGATAAAAAAGAACCTCTGCTCAACAAGTTCCAGAT TACAACTTCACCAGGTTCAACTCAAAAAATATTAACAGCAATGATTGGGTTAAATAACAAAACATTAGAC GATAAAACAAGTTATAAAATCGATGGTAAAGGTTGGCAAAAAGATAAATCTTGGGGTGGTTACAACGTTA CAAGATATGAAGTGGTAAATGGTAATATCGACTTAAAACAAGCAATAGAATCATCAGATAACATTTTCTT TGCTAGAGTAGCACTCGAATTAGGCAGTAAGAAATTTGAAAAAGGCATGAAAAAACTAGGTGTTGGTGAA GATATACCAAGTGATTATCCATTTTATAATGCTCAAATTTCAAACAAAAATTTAGATAATGAAATATTAT TAGCTGATTCAGGTTACGGACAAGGTGAAATACTGATTAACCCAGTACAGATCCTTTCAATCTATAGCGC ATTAGAAAATAATGGCAATATTAACGCACCTCACTTATTAAAAGACACGAAAAACAAAGTTTGGAAGAAA AATATTATTTCCAAAGAAAATATCAATCTATTAACTGATGGTATGCAACAAGTCGTAAATAAAACACATA AAGAAGATATTTATAGATCTTATGCAAACTTAATTGGCAAATCCGGTACTGCAGAACTCAAAATGAAACA AGGAGAAACTGGCAGACAAATTGGGTGGTTTATATCATATGATAAAGATAATCCAAACATGATGATGGCT ATTAATGTTAAAGATGTACAAGATAAAGGAATGGCTAGCTACAATGCCAAAATCTCAGGTAAAGTGTATG ATGAGCTATATGAGAACGGTAATAAAAAATACGATATAGATGAATAA

Briefly, an RNP1 was preassembled with a gRNA sequence designed to target MRSA DNA. Specifically, RNP1 was designed to target a 20 bp region of the mecA gene of MRSA: TGTATGGCATGAGTAACGAA (SEQ ID NO: 8). An RNP2 was preassembled with a gRNA sequence designed to target an unblocked nucleic acid molecule that results from unblocking (i.e., linearizing) a circularized blocked nucleic acid molecule. The circularized blocked nucleic acid molecule was designed and synthesized (SEQ ID NO: 6): GTT*AT*TA*AA*TG*AC*TT*CT*CATT, where the * indicate bonds that are phosphorothioate modified. The 5′ and 3′ ends were covalently linked to form a circularized molecule. MRSA DNA (SEQ ID NO: 7) with 3000, 300, 30, or 3 total copies, or a negative control (e.g., 0 copies), were added to a reaction mixture to begin the cascade assay. The reaction mix contained the preassembled RNP1, preassembled RNP2, and a circularized blocked nucleic acid molecule, in a buffer (pH of about 8) containing 4 mM MgCl₂ and 101 mM NaCl. The buffering conditions were optimized to reduce non-specific nickase activity by the RNP complexes. The cascade assay proceeded for 10 minutes at 37° C., and fluorescence from the reporter moiety was measured. In all titrations, a significant change in fluorescence was observed after 10 and 5 minutes, relative to the negative control (see the results in FIG. 8 ). The cascade assay was initiated to identify the presence of MRSA in 10 minutes or 5 minutes at 37° C. Data represent 3 independent biological replicates. Data is presented as mean±s.d.****=p<0.0001 (student t-test). The results indicate that the cascade assay can detect as few as 3 MRSA target molecules in only 5 minutes when at 37° C.

Example VIII: Detection of MRSA in Under 10 Minutes with a Cascade Assay at 25° C.

To detect the presence of MRSA and determine the sensitivity of detection with the cascade assay, titration experiments with MRSA DNA (SEQ ID NO: 7) were performed.

Briefly, an RNP1 was preassembled with a guide RNA (gRNA) sequence designed to target MRSA DNA. Specifically, RNP1 was designed to target the following 20 bp sequence in the mecA gene of MRSA: TGTATGGCATGAGTAACGAA (SEQ ID NO: 8). An RNP2 was preassembled with a gRNA sequence designed to target an unblocked nucleic acid molecule that results from unblocking (i.e., linearizing) a circularized blocked nucleic acid molecule. A circularized blocked nucleic acid molecule was designed and synthesized (SEQ ID NO: 6): GTT*AT*TA*AA*TG*AC*TT*CT*CATT, where the * indicate bonds that are phosphorothioate modified. The 5′ and 3′ ends were covalently linked to form a circularized molecule.

MRSA DNA (SEQ ID NO: 7) with 30000, 3000, 300, 30, or 3 total copies, or a negative control (e.g., 0 copies), was added to a reaction mixture to begin the cascade assay. The reaction mix contained the preassembled RNP1, preassembled RNP2, the circularized blocked nucleic acid molecule in a buffer (˜pH 8) containing 4 mM MgCl₂ and 101 mM NaCl. The buffering conditions were optimized to reduce non-specific nickase activity by the RNP complexes. The cascade reaction proceeded for 20 minutes at 25° C., and fluorescence by the reporter molecule was measured. In all titrations, a significant change in fluorescence was observed after 10 and 5 minutes, relative to the negative control (see the results in FIG. 9 ), indicating that the cascade assay can detect as few as 3 MRSA target molecules in 10 minutes or less while at room temperature. The data represent 3 independent biological replicates and is presented as mean±s.d.****=p<0.0001 (student t-test).

Example IX: Optimized Detection of MRSA in 1 Minute with the Cascade Assay at 25° C.

RNP1 was preassembled with a gRNA sequence designed to target MRSA DNA (SEQ ID NO: 7). Specifically, RNP1 was designed to target the following 20 bp sequence in the mecA gene of MRSA: TGTATGGCATGAGTAACGAA (SEQ ID NO: 8). RNP2 was preassembled with a gRNA sequence designed to target an unblocked nucleic acid molecule that results from unblocking a blocked nucleic acid molecule. Five different double stranded and linear blocked nucleic acid molecules were designed, synthesized, and tested: molecule C5, molecule C6, molecule C7, molecule C8, and molecule C9. The nucleotide sequences of molecules C5-C9 are as follows.

C5 (SEQ ID NO: 9): GTTATTGAGAATTATTGTCATATTATTCTAATATTATTAAGGCTTATT CACTGTTATTATTATAATTATTAAGCTTATT C6 (SEQ ID NO: 10): GTTATTGAGAAGTTATTATCATCTATTATTAATAAGTTATTGCCACTA TTATTGTTATAATTATTAAGCTTATT C7 (SEQ ID NO: 11): GTTATTGAGAAGTATTATTCATCTAATTATTATAAGGCCTTATTACTG TTATTATTAATAAGCTTATT C8 (SEQ ID NO: 12): GTTATTGAGAAGTCTTATTATCTAATATTATTAGGCCACTGTTATTAT TATAATAAGCTTATT C9 (SEQ ID NO: 13): GTTATTGAGAAGTCATTATTATCTAATAAGTTATTGCCACTGTTATTA TTATAATAAGCTTATT

Three copies of MRSA DNA (SEQ ID NO: 7) or a negative control (e.g., 0 copies) were added to a reaction mix to begin the cascade assay. The reaction mix contained the preassembled RNP1, preassembled RNP2, and one of the five blocked nucleic acid molecules in a buffer (˜pH 8) containing 4 mM MgCl₂ and 71 mM NaCl. These buffering conditions were optimized to reduce non-specific nickase activity by the RNP complexes. Each cascade assay proceeded for 10-20 minutes at 25° C., and fluorescence by the reporter molecule was measured for each cascade assay containing C5 (see the results shown in FIG. 10 , where the presence of just 3 MRSA targets was detected in 5 minutes or less at 25° C. The data represent 9 independent biological replicates and is presented as mean±s.d.****=p<0.0001 (student t-test), molecule C6 (see the results shown in FIG. 11 , where the presence of just 3 MRSA targets was detected in 5 minutes or less at 25° C. The data represent 6 independent biological replicates and is presented as mean±s.d.****=p<0.0001 (student t-test)), molecule C7 (see the results shown in FIG. 12 , where the presence of just 3 MRSA targets was detected in 5 minutes or less at 25° C. Data represent 6 independent biological replicates and is presented as mean±s.d.****=p<0.0001 (student t-test)), molecule C8 (see the results shown in FIG. 13 , where the presence of just 3 MRSA targets was detected in 5 minutes or less at 25° C. Data represent 6 independent biological replicates and is presented as mean±s.d.****=p<0.0001 (student t-test)), and molecule C9 (see the results shown in FIG. 14 , where the presence of just 3 MRSA targets was detected in 10 minutes or less at 25° C. Data represent 6 independent biological replicates and data is presented as mean±s.d.****=p<0.0001 (student t-test)). A significant change in fluorescence is observed after 1 minute and after 5 minutes, relative to the negative control, indicating that the cascade assay can be optimized to detect as few as 3 MRSA target molecules in as little as 1 minute while at room temperature.

While certain embodiments have been described, these embodiments have been presented by way of example only and are not intended to limit the scope of the present disclosures. Indeed, the novel methods, apparatuses, modules, instruments and systems described herein can be embodied in a variety of other forms; furthermore, various omissions, substitutions and changes in the form of the methods, apparatuses, modules, instruments and systems described herein can be made without departing from the spirit of the present disclosures. The accompanying claims and their equivalents are intended to cover such forms or modifications as would fall within the scope and spirit of the present disclosures. 

We claim:
 1. A reaction mixture for a CRISPR nuclease cascade assay comprising: a first ribonucleoprotein (RNP) (RNP1) complex comprising a first CRISPR nuclease and a first guide RNA (gRNA); wherein the first gRNA comprises a sequence complementary to a target nucleic acid of interest, and wherein the first CRISPR nuclease nuclease exhibits both cis-cleavage activity and trans-cleavage activity; a second ribonucleoprotein (RNP2) complex comprising a second CRISPR nuclease and a second gRNA that is not complementary to the target nucleic acid of interest; wherein the second CRISPR nuclease exhibits both CRISPR nuclease; and a plurality of blocked nucleic acid molecules comprising a sequence complementary to the second gRNA, wherein the blocked nucleic acid molecules do not bind to the RNP1 complex or the RNP2 complex.
 2. The reaction mixture of claim 1, wherein the first and/or second CRISPR nuclease is a Cas3, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas14, Cas12h, Cas12i, Cas12j, Cas13a, Cas13b nuclease.
 3. The reaction mixture of claim 1, wherein the first CRISPR nuclease is a different CRISPR nuclease than the second CRISPR nuclease.
 4. The reaction mixture of claim 1, wherein the first and/or second CRISPR nuclease is a Type V or Type VI CRISPR nuclease.
 5. The reaction mixture of claim 1, wherein the first and/or second CRISPR nuclease comprises a RuvC nuclease domain or a RuvC-like nuclease domain and lacks an HNH nuclease domain.
 6. The reaction mixture of claim 1, wherein the blocked nucleic acid molecules comprise a structure represented by any one of Formulas I-IV, wherein Formulas I-IV comprise in the 5′-to-3′ direction: (a)A-(B-L)_(J)-C-M-T-D  (Formula I); wherein A is 0-15 nucleotides in length; B is 4-12 nucleotides in length; L is 3-25 nucleotides in length; J is an integer between 1 and 10; C is 4-15 nucleotides in length; M is 1-25 nucleotides in length or is absent, wherein if M is absent then A-(B-L)_(J)-C and T-D are separate nucleic acid strands; T is 17-135 nucleotides in length and comprises at least 50% sequence complementarity to B and C; and D is 0-10 nucleotides in length and comprises at least 50% sequence complementarity to A; (b)D-T-T′-C-(L-B)_(J)-A  (Formula II); wherein D is 0-10 nucleotides in length; T-T′ is 17-135 nucleotides in length; T′ is 1-10 nucleotides in length and does not hybridize with T; C is 4-15 nucleotides in length and comprises at least 50% sequence complementarity to T; L is 3-25 nucleotides in length and does not hybridize with T; B is 4-12 nucleotides in length and comprises at least 50% sequence complementarity to T; J is an integer between 1 and 10; A is 0-15 nucleotides in length and comprises at least 50% sequence complementarity to D; (c)T-D-M-A-(B-L)_(J)-C  (Formula III); wherein T is 17-135 nucleotides in length; D is 0-10 nucleotides in length; M is 1-25 nucleotides in length or is absent, wherein if M is absent then T-D and A-(B-L)_(J)-C are separate nucleic acid strands; A is 0-15 nucleotides in length and comprises at least 50% sequence complementarity to D; B is 4-12 nucleotides in length and comprises at least 50% sequence complementarity to T; L is 3-25 nucleotides in length; J is an integer between 1 and 10; and C is 4-15 nucleotides in length; or (d)T-D-M-A-L_(p)-C  (Formula IV); wherein T is 17-31 nucleotides in length (e.g., 17-100, 17-50, or 17-25); D is 0-15 nucleotides in length; M is 1-25 nucleotides in length; A is 0-15 nucleotides in length and comprises a sequence complementary to D; and L is 3-25 nucleotides in length; p is 0 or 1; C is 4-15 nucleotides in length and comprises a sequence complementary to T.
 7. The reaction mixture of claim 6, wherein: (a) T of Formula I comprises at least 80% sequence complementarity to B and C; (b) D of Formula I comprises at least 80% sequence complementarity to A; (c) C of Formula II comprises at least 80% sequence complementarity to T; (d) B of Formula II comprises at least 80% sequence complementarity to T; (e) A of Formula II comprises at least 80% sequence complementarity to D; (f) A of Formula III comprises at least 80% sequence complementarity to D; (g) B of Formula III comprises at least 80% sequence complementarity to T; (h) A of Formula IV comprises at least 80% sequence complementarity to D; and/or (i) C of Formula IV comprises at least 80% sequence complementarity to T.
 8. The reaction mixture of claim 1, wherein the blocked nucleic acid molecules comprise a first sequence complementary to the second gRNA and a second sequence not complementary to the second gRNA, wherein the second sequence at least partially hybridizes to the first sequence resulting in at least one loop.
 9. The reaction mixture of claim 1, wherein the reaction mixture comprises about 1 fM to about 10 μM of the RNP1.
 10. The reaction mixture of claim 1, wherein the reaction mixture comprises about 1 fM to about 1 mM of the RNP2.
 11. The reaction mixture of claim 1, wherein the reaction mixture comprises at least two different RNP1s, wherein different RNP1s comprise different gRNA sequences.
 12. The reaction mixture of claim 11, wherein the reaction mixture comprises 2 to 10000 different RNP1s.
 13. The reaction mixture of claim 12, wherein the reaction mixture comprises 2 to 1000 different RNP1s.
 14. The reaction mixture of claim 13, wherein the reaction mixture comprises 2 to 100 different RNP1s.
 15. The reaction mixture of claim 14, wherein the reaction mixture comprises 2 to 10 different RNP1 complexes.
 16. The reaction mixture of claim 1, wherein the blocked nucleic acid molecules include the sequence of any one of SEQ ID NOs: 14-1421.
 17. The reaction mixture of claim 1, wherein the blocked nucleic acid molecules are circular.
 18. The reaction mixture of claim 1, wherein the blocked nucleic acid molecules are linear.
 19. The reaction mixture of claim 1, wherein a K_(d) of the blocked nucleic acid molecules to the RNP2 is about 10⁵-fold greater or more than the K_(d) of unblocked nucleic acid molecules.
 20. The reaction mixture of claim 1, wherein the RNP2 complex recognizes a PAM sequence.
 21. The reaction mixture of claim 1, wherein the RNP2 complex does not recognize a PAM sequence.
 22. The reaction mixture of claim 1, wherein the target nucleic acid of interest is of bacterial, viral, fungal, mammalian or plant origin.
 23. The reaction mixture of claim 1, further comprising a reporter moiety: wherein the reporter moiety comprises a DNA, RNA or chimeric nucleic acid molecule and is operably linked to the blocked nucleic acid molecule that produces a detectable signal upon cleavage by RNP1 and/or RNP2; or wherein the reporter moiety comprises a DNA, RNA or chimeric nucleic acid molecule and is not operably linked to the blocked nucleic acid molecule and produces a detectable signal upon cleavage by RNP1 and/or RNP2.
 24. The reaction mixture of claim 23, wherein the detectable signal is produced within about 1-10 minutes upon binding of the target nucleic acid of interest to RNP1.
 25. The reaction mixture of claim 24, wherein the detectable signal is a fluorescent, chemiluminescent, radioactive, colorimetric or other optical signal.
 26. The reaction mixture of claim 24, wherein the reporter moiety comprises a modified nucleoside or nucleotide.
 27. The reaction mixture of claim 26, wherein the modified nucleoside or nucleotide comprises a locked nucleic acid (LNA), peptide nucleic acid (PNA), 2′-O-methyl (2′-O-Me) modified nucleoside, 2′-fluoro (2′-F) modified nucleoside, and/or a phosphorothioate (PS) bond.
 28. The reaction mixture of claim 1, wherein the blocked nucleic acid molecule is a blocked primer molecule. 